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Immunological and molecular characterization of the STX-10 antigen Hsiao, Letticia 1994

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IMMuNOLOGICAL AND MOLECULAR CHARACTERIZATION OF THE STX-lO ANTIGEN  By Letticia Hsiao  B.Sc., The University of Victoria, 1991  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER IN SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY REPRODUCTIVE AND DEVELOPMENTAL SCIENCES PROGRAM  We accept this thesis as conforming to the required standard  UNIVERSITY OF BRITISH COLUMBIA. 1994 Øetticia Hsiao, 1994  In presenting this thesis partial in fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted the by head of my department or his by or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of__________________ The University of Briti Vancouver, Canada Date9/94 17  7,  Columbia  ABSTRACT  The STX-10 antigen, recognized by the monoclonal antibody HSA-10, was found to be present in human placenta and in human sperm.  This antigen was isolated from  placenta and sperm extracts by immunoaffinity chromatography, it was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by western blotting to be a 75±5 kD protein. By enzyme-linked immunosorbent assay, the isolated protein was found  to react specifically with rabbit anti-STX- 10 serum and with HSA- 10 antibody.  The  immunoreactivity of the STX- 10 protein was quantitatively determined by sandwich enzyme immunosorbent assay, with HSA-10 as the capturing and detecting antibody. By indirect immunofluorescent assay, the STX-10 antigen in sperm was found to be localized to the inner acrosome of human sperm. Rabbit anti-STX-10 sera were used to screen a Xgtl 1 human placenta eDNA library. Following isolation and purification of positive cDNA clones, the cDNA inserts were analyzed by restriction enzyme digestions, and PCR amplifications. One clone, 1 kb in size, was obtained. The cDNA insert of the 1 kb clone was sequenced and found to be mainly composed of the 3’untranslated region, including an mR.NA degradation signal. In the 3’-untranslated region, an identical sequence was found to 411 bp of a 449 bp clone isolated from the male liver HepG2 carcinoma cell line. The 1 kb cDNA clone contained a possible open reading frame of 168 amino acids in size. The full length of the open reading frame for the STX-10 gene still remains to be elucidated. In Northern blots, the 1 kb cDNA fragment was used as the probe. The mRNA derived from human carcinoma BeWo cell line, known to express the STX-10 antigen, was found to be 5.5 kb in length. In conclusion, the STX- 10 antigen which is recognized by monoclonal antibody HSA- 10, is an unique antigen that should be strongly considered for the development of an immunocontraceptive vaccine.  II  TABLE OF CONTENTS  Page Abstract  .  .  .  List of Figures. List of Abbreviations Acknowledgements  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  ii v vi viii  .  Introduction  1  Background The use of monoclonal antibodies to study sperm antigens Characterization of sperm antigens  2 4  Monoclonal antibodies as probes to study sperm physiological changes Characterization of the STX-10 antigen .  10 12  Materials and Methods Chemicals  14  Source of Antibodies Sources of Human Placenta and Sperm Extracts Antigen Purification by Immuno affinity Chromatography  .  16  Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Western Blot Assay Enzyme-linked Immunosorbent Assay (ELISA) .  .  .  .  19  .  20  .  21  Immunoscreening of Human Placenta cDNA Library. Isolation and Purification of cDNA Clones PCR Amplification of cDNA Agarose Gel Electrophoresis  23 24  .  26  .  .  111  17 18  .  Sandwich Enzyme Immunoassay (SEIA) Indirect Immunofluorescent Assay  15 15  .  .  26  Western Blot of Fusion Proteins  27  DNA Sequencing  29  Isolation of RNA  30  Northern Blot  31  Purification and Characterization of the STX-10 Antigen Enzyme-linked Immunosorbent Assay  32  Sandwich Enzyme Immunoassay  37  Localization of STX- 10 Antigen by Indirect Immunofluorescent Assay Immunoscreening of Human Placenta cDNA Library.  40  Western Blot of Plate Lysates Obtained from cDNA Clones  47  Analysis of DNA Isolated from Positive Clones  50  Northern Blot Analysis  59  Results 37  46  Discussion  62  Conclusions  67  References  68  Appendix A  82  Appendix B  82  iv  LIST OF FIGURES Figure 1.  Elution profile of the STX-10 antigen purified by an HSA-10 immunoaffinity column.  33  Sodium dodecylsulfate polyacrylamide gel electrophoresis and Western Blots of the purified STX-10 protein.  35  Sandwich enzyme immunoas say with HSA- 10 Mab as the coated antibody on wells and as the detecting antibody.  38  .  Figure 2.  .  .  .  .  Figure 3.  Figure 4.  .  Indirect immunofluorescence localization of STX- 10 antigen on methanol-fixed human sperm.  42  Time-dependent changes of HSA-10 Mab binding to STX-10 Ag on human sperm as detected by indirect immunofluorescent assay.  44  Figure 6.  Western blot of fusion proteins expressed by isolated cDNA clones.  48  Figure 7.  Possible open reading frames for the 1 kb cDNA sequence.  52  Figure 8.  Nucleotide sequence of the placenta STX- 10 1 kb cDNA clone and deduced amino acid sequence.  54  Figure 9.  Kyte-Doolittle hydropathy plot of the deduced amino acid sequence  57  Figure 10.  Northern Blot of BeWo cells and mouse testes probed with radioactively-labelled 1 kb cDNA fragment.  60  .  Figure 5.  .  .  .  .  V  .  .  .  .  .  .  LIST OF ABBREVIATIONS  Ab Ag ALP  Antibody Antigen  BLAST  Basic alignment search tool  bp  Base pairs  BSA cDNA  Bovine serum albumin Complementary deoxyribonucleic acid  DAB  Diaminobenzidine  DEPC O 2 dH  Diethyl pyrocarbonate Deionized water  DMSO  Dimethyl sulfoxide  DNA  Deoxyribonucleic acid  dNTPs DTT  Deoxy nucleotide triphosphates Dithiothreitol  ECL  Enhanced chemiluminenscence  E. coli  Escherichia coil  EDTA  Ethylenediamine Tetraacetic Acid  ELISA  Enzyme linked immunosorbent assay  FITC  Fluorescein Isothiocyanate  PdATP 32 y  32 GammaP-labelled-deoxyadenosine triphosphate  HKP  Horseradish peroxidase  HSA-1O IPTG  Human sperm antibody-b Indirect immunofluorescent assay Isopropylthio-3-galactoside  kD  Kilodaltons  IFA  Alkaline phosphatase  vi  LB Broth  Luria-Bertani Broth  LDB  Lamba Dilution Buffer  Mab  Monoclonal antibody  mR.NA  Messenger ribonucleic acid  NRS  Normal rabbit serum  O.D.  Optical density  Pab  Polyclonal antibody  PBS PCR  Phosphate buffered saline Polymerase Chain ReactionTM, Perkin-Elmer Cetus  PEG  Polyethylene glycol  pfu/ml  Plaque forming units per milliliter  RNA  Ribonucleic acid  rpm  revolutions/rotations per minute  SDS-PAGE  Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis  SEJA  Sandwich enzyme immunoassay  STX- 10 TAE  Sperm/placenta cross-reacting antigen- 10 Tris-acetate/EDTA electrophoresis buffer  TBS  Tris-buffered saline  TBST TE buffer  Tris-buffered saline with Tween-20 Tris-EDTA buffer  TIVIB  3,3-5, 5’-tetramethylbenzidine  TMS  Thimerosal  UV light  Ultraviolet light  vii  ACKNOWLEDGEMENTS  To my committee consisting of Dr. Lee, Dr. Moon, Dr. Rajamahendran, Dr. Skala, and Dr. Gillam, I would like to thank each of you for your support and advice, especially my chairman, Dr. Young Suk Moon, and my supervisor, Dr. Gregory Chi-Yu Lee.  To Biao-Yang Lin, thank you for your expertise and help with molecular biology techniques. To Dr. Eric Wong and Dr. Yang, thank you for all your help and assistance with my thesis project.  To Dr. Yoshiki and Divarkar Ambrose, thank you both for your invaluable knowledge, expertise, love of science, and friendship.  To Margaret, Andrew, Logi, and Pat, thank you for your advice and for always being there. To Pat, fellow student and cave-dweller, thank you for travelling this road together with me. Your input, your friendship, our Blue Chip ventures, and your understanding.will always be deeply appreciated.  To my family and friends-especially my parents, Karen, Ken, and Nicky- thank you for your patience, love, and caring over the past two years. I would not have been able to make it through without your advice, support and understanding.  “iii  INTRODUCTION  Immunocontraception is the idea of an immunological method of contraception by the production of an immune response in the body that prevents, blocks, or disrupts fertility. This is generally done by immunization of antigens to interfere with gamete interactions by preventing sperm transport and blocking fertilization or by inhibiting or disrupting implantation. The antigens of greatest interest are: sperm antigens, zona pellucidaloocyte antigens, and hormonal antigens-like human chorionic gonadotropin (hCG) (Griffin, 1991). Antigens can be peptides, proteins, or any foreign substances which induce an immune response in the body. This response may be cellular, with T-cell mediation, or the response may be humoral, with the production of a specific antibody (Benjamini and Leskowitz, 1991). Since antigens can be recognized by antibodies, antibodies may initially be used to isolate, purify, and characterize the antigen.  Monoclonal antibodies and polyclonal antibodies are used to recognize antigens. Monoclonal antibodies are derived from a single clone and therefore, they have a single specificity and recognize a specific epitope of a protein (Benjamini and Leskowitz, 1991). The cell fusion of an antibody-secreting B-cell and a myeloma tumour cell results in a hybrid cell which proliferates continuously and secretes a single immunoglobulin. Monoclonal antibodies have well-defined specificities because they recognize only one antigenic determinant (Benjamini and Leskowitz, 1991).  I  Polyclonal antibodies are  essentially composed of a mixture of numerous antibodies with different immunoglobulin classes, different structures and different epitope specificities.  The idea of immunocontraception is not a new one. Nearly one hundred years ago, in 1899, Landsteiner and Metchnikoff independently showed that the injection of spermatazoa from one species into a different species produced an antibody response (Landsteiner, 1899; Metchnikoff, 1899).  However, whole sperm should not be used to develop a  vaccine since many sperm antigens are shared or cross-react with somatic tissues (Naz, 1988).  In the 1970’s, sperm-specific enzymes of the acrosome, such as, acrosin,  hyaluronidase, and lactate dehydrogenase, were produced in large quantities and used to immunize animals; however, for acrosin and hyaluronidase, both active and passive immunizations did not significantly reduce fertility (Syner et al., 1979; Morton and McAnulty, 1979). In the 1990’s, specific epitopes are being characterized in a wide variety of species. The most effective immunocontraceptive will likely involve a combination of either different antigens or different antibodies to interfere with the process of fertilization at the various stages.  These gamete-specific antigens have the distinct advantage of  preventing fertilization rather than disrupting implantation like the hCG vaccine (Jones, 1992).  2  BACKGROUND  THE USE OF MONOCLONAL ANTIBODIES TO STUDY SPERM ANTIGENS  Since the development of hybridoma technology by K5hler and Milstein in 1975, monoclonal antibodies have been widely used in many fields of scientific research (Hurrell, J. ed., 1981; Fellows, R.E. and Eisenbarth, G.S., eds., 1981).  Using monoclonal  antibodies, the knowledge of specific antigens expressed within the mammalian reproductive systems has greatly advanced (Isojima, 1990; Naz, 1990, Alexander, 1989). Monoclonal antibodies have several advantages over the conventional polyclonal antibodies in terms of specificity, homogeneity, perpetuity, and availability (Benjamini and Leskowitz, 1991; Smith, 1992). These characteristics have led to the development of several different immunoassay techniques including: indirect immunofluorescence, immunocytochemistry, immunoaffinity chromatography, and enzyme-linked immunosorbent assays. In our research laboratory, monoclonal antibodies (Mabs) against sperm antigens were generated by immunizing mice with human sperm.  Monoclonal antibody (Mab)  HSA- 10 was generated by immunizing BALB/c mice with acrosome-reacted human sperm as previously reported by Lee et. al. (1993). This antibody was evaluated by the World Health Organization (WHO) sperm antigen workshop II and sperm antigen nomenclature project in 1990.  HSA-10 Mab was designated as S75 and found to react with only  acrosome-reacted human sperm, human placenta/trophoblast and mouse oocytes/embryos (Anderson, 1990; Lee et al., 1993; Polgar et al., 1994). This antibody was placed on the 3  high priority list as a candidate for immunocontraceptive vaccine development based on (a) its high specificity to human sperm antigen; (b) its lack of cross-reactivity to any other somatic tissue; and (c) its significant inhibition of in vitro fertilization and other sperm functions. Sperm functions are generally evaluated in vitro by such assays as the zona-free hamster egg penetration test (Yanagimachi et al., 1976), cervical mucus penetration test (Kremer and Jager, 1976), complement-dependent immobilization studies (Isojima et a!., 1972; Koyama et al., 1988) and agglutination studies (Friberg, 1974; Jager et al., 1978; Mathur et a!, 1979).  CHARACTERIZATION OF SPERM ANTIGENS  Monoclonal antibodies have been used to study sperm-specific antigens in several species. These antigens have been studied more extensively using molecular biology and recombinant DNA techniques. With these techniques, large amounts of the antigens can be produced, structure-function relationships between the antigen and other components can be analyzed, and their physiological roles can be further defined. Using a combination of these techniques, a variety of sperm antigens have been characterized (Isojima, 1990; Naz, 1990a,b; Wang and Heap, 1992). These include plasma membrane antigens, acrosomal antigens, sperm-coating antigens, cytoplasmic, mitochondrial, and nuclear antigens in various species (Wang and Heap, 1992). The characterizations of some of these antigens  4  by other researchers in the same field will be discussed.  Recombinant DNA technology involves the incorporation of a foreign gene into a unique site of a plasmid or lambda phage.  The plasmid or phage will express its proteins,  including the foreign gene, as a fusion protein. A compatible strain of Escherichia coil bacteria is transfected with the phage or plasmid. As the recombinant protein is produced, the bacterial host is lysed. There are limited natural sources of human sperm and other human tissues; therefore, recombinant DNA technology has been used extensively for the production of recombinant human proteins. DNA technology has greatly enhanced and advanced the knowledge of sperm structure and function. As sperm antigen contraceptive vaccines are developed, this technology will continue to play an exclusive role.  Rabbit sperm antigens (RSA) have been studies since 1976 by O’Rand and his group (O’Rand, 1981; O’Rand et al., 1984, 1988, 1989). This family of low molecular weight proteins (10-13 kDs and 14-18 kDs) are found on the equatorial segment, the post acrosome and midpiece of rabbit sperm (O’Rand and Widgren, 1989). A Mab raised to RSA-1 was found to inhibit in vitro fertilization and possibly played a role in sperm-zona binding (O’Rand, 1984). It was noted that this Mab also cross-reacted with RSA-2 and RSA-3 antigens and to human sperm extract (ORand, 1988).  However, the antigen in  human sperm was localized to the middle region of the tail unlike in rabbit sperm, where the antigen was localized to the equatorial region (Esaguy et al., 1988). More recently, the  14 kD protein was digested with Kallikrein and analysed by HPLC. A peptide of 10 amino acids, termed P1OG, was found to block sperm binding to zona pellucida.  Anti-sera  generated against P1 OG was found to react with rabbit sperm at the equatorial region (O’Rand, 1989).  , lactate dehydrogenase-C 4 LDH-C , is a sperm specific enzyme that has been well 4 characterized by Goldberg et al. (Goldberg, 1963; Blanco and Zinkman, 1963).  This  enzyme is localized mainly on the tail of mouse sperm and on the acrosome of human sperm (Hintz and Goldberg, 1977; Wu et al., 1987).  Although there are other LDH  isozymes in various tissues throughout the body, LDH-C 4 and the other isozymes are immunologically distinct (Goldberg, 1990). Antibodies to this enzyme were found to (1) suppress fertility in female mice, rabbits and baboons (Goldberg, 1975; Wheat and Goldberg, 1983); and to (2) agglutinate mouse, rabbit, and human sperm (Goldberg, 1990). When female baboons were actively immunized with mouse LDH-C , a 70% reduction in 4 fertility was observed (Goldberg and Shelton, 1986). The human LDH-C 4 gene has been sequenced, and the genomic structure and promoter activity of the gene has been elucidated (Cooker et al., 1993).  SP-10 antigen has been well characterized by John Herr and his group, using the MHS-10 monoclonal antibody (Herr et al., 1989; Herr et al., 1990a,b; Wright et al., 1990; Kurth et al., 1991, 1993; Foster and Herr, 1992; Wright et al., 1993, Freemerman et al., 1993,  6  1994). Using this antibody, SP-10 was found to be localized on the acrosomal region of human sperm (Herr et al., 1989).  During spermatogenesis, SP-l0 was found to be  expressed by only the round spermatids and the ensuing stages of spermiogenesis (Kurth et al., 1993). In mature sperm, SP-10 is an intra-acrosomal protein of 24-34 kD that remains associated with the inner acrosomal membrane and the equatorial segment after the acrosome reaction occurs (Herr et al., 1990; Kurth et al., 1991). The human SP-l0 amino acid sequence showed 60-85% homology with macque, baboon, and mouse SP-10 sequences (Freemerman et al., 1993; Liu et al., 1992).  PH-20 and PH-30 antigens from guinea pig have been studied by Primakoff since 1983 (Primakoffet al., 1983, 1985, 1988a,b, 1990). The PH-20 antigen, which is involved in the adhesion of sperm to the zona pellucida, is localized on the sperm plasma membrane and the acrosomal membrane (Primakoff et al., 1985; Myles et al., 1987).  Both the Mab  against PH-20 and anti-PH-20 polyclonal sera showed 80-94% inhibition to the binding of acrosome-reacted sperm to zona pellucida, as compared to controls (Primakoff et al., 1985, 1988). While the Mab to PH-20 reacted only to guinea pig sperm acrosome, the polyclonal antisera was found to react with guinea pig and human sperm (Primakofl 1988). Using purified PH-20 as the immunogen, female guinea pigs were sterile for six to fifteen months.  Also, the length of immunization (in days) varied between individual  guinea pigs but was found to be completely reversible (Primakoff et al., 1988).  From  Southern blot analysis, the PH-20 gene was found to be present in mouse, rat, hamster,  7  rabbit, bovine, monkey and human genomic DNA (Lathrop et a!., 1990). The full-length cDNA sequence was obtained and found to be a new protein with no significant homology to any other known proteins.  The PH-30 antigen, localized on the sperm surface, consists of 2 subunits, c and j3. Although this antigen is essential for the fusion of sperm and egg, active immunization with PH-30 only partially reduced fertility (Blobel et a!., 1992).  MSA-63, a mouse sperm acrosomal antigen has been well studied by Liu and Lee (Liu et al., 1989; Lee et al., 1990; Liu et al., 1992). cDNA clones expressing recombinant MSA 63 antigen were isolated from a mouse testes cDNA library and then expressed in E. coil. Female mice were immunized and the resulting antisera were found to react only with the sperm acrosome. The antisera also significantly inhibited in vitro fertilization of mouse oocytes (Liu et al., 1990) and inhibited human sperm penetration of zona-free hamster eggs (Liu et al., 1989).  However, passive immunizations with HS-63 Mab and polyclonal  antibody against MSA-63 did not dramatically reduce in vivo fertility although the results were statisically significant (Liu MS, Ph.D. thesis, 1991). The purified MSA-63 antigen was also recognized by HS-1 1 Mab and HSK-9 Mab (Ambrose et a!., in press).  Similar to HS-63 Mab, HS-1 1 reacted specifically with the  acrosomal region of sperm from several mammalian species. These two antibodies did not cross-react with human, mouse or bull somatic tissues. In mouse testis, HS-63 was found  8  to react with a group of proteins between 27-70 kD. HS-11 was found to react to a group of membrane-associated antigens between 3 5-50 kD (Lee et a!., 1990). Both antibodies were found to inhibit fertilization by interfering with sperm-zona binding and with zona induced acro some reaction (Fann and Lee, 1992). At the nucleotide/protein level, MSA-63 was found to have a high degree of homology to the SP-10 antigen from human sperm (Liu et a!., 1992). Although there is a high degree of homology, neither Mab is capable of recognizing the other antigen. Therefore, these two antigens are immunologically distinct and possess different epitopes. The epitope recognized by HS-63 Mab is species-conserved whereas the epitope recognized by MHS- 10 Mab is specific to human sperm. It has been established that the mouse MSA-63 protein is a homolog of human SP-10. It is presently unknown if these two intra-acrosomal antigens share the same biological functions (Liu et al., 1992).  Despite the extensive research efforts carried out by these researchers, none of these sperm antigens have been fully characterized for human application as effective contraceptive vaccines. Nevertheless, through the basic molecular studies of selected sperm antigens, significant advances have been made regarding the structure/function of sperm antigens and their roles during the fertilization process.  9  MONOCLONAL ANTIBODIES AS PROBES TO STUDY SPERM MEMBRANE CHANGES  The two most important physiological processes associated with sperm prior to fertilization are capacitation and the acrosome reaction.  The process of capacitation, which is an  asynchronous one, must be completed before the acrosome reaction can occur. During capacitation, several changes occur (Storey, 1991). Certain seminal plasma and epidydimal proteins that were absorbed or coated on the sperm surface (during maturation in the epididymis) are now removed (Isojima, 1990). Cholesterol, which acts as a membrane stabilizer, is removed (Zaneveld et al., 1991). This results in the rearrangement of the membrane proteins in the lipid bilayer. The sperm head consists of three membranes: the inner acrosomal membrane, the outer acrosomal membrane, and the plasma membrane. During the acrosome reaction, the outer acrosomal membrane and the plasma membrane fuse together at various points.  As the calcium concentration increases, the joined  membranes start to swell and finally burst. The acrosomal contents, including acrosin and hyaluronidase and many other components, are exposed and the inner acrosomal membrane now externalized.  In mouse sperm, the acrosome reaction takes place after the initial  binding of the sperm to the zona pellucida (Myles and Primakoff, 1991). In human sperm, it is still unknown whether the acrosome reaction takes place before or after binding to the zona pellucida.  Carbohydrate-protein interactions result in sperm recognition and  attachment to the zona pellucida (Alexander, 1989). The zona pellucida, specifically the  I0  ZP3 glycoprotein, initially binds to the sperm head and induces the acrosome reaction (Florman and Wassarman, 1985; Wassarman, 1988). Secondary binding occurs between the ZP2 glycoprotein of the zona pellucida and acrosome-reacted sperm. This is followed by the fusion of the sperm and egg plasma membranes.  Since Mabs recognize different sperm antigens at various stages during the fertilization process, they can be used to identify and monitor these stages. For example, the Mab S60 (also known as H3 16) is a good marker of the acrosome reaction because it does not react to acrosome-intact sperm, it can be used on unfixed viable cells, and it binds strongly to the inner acrosomal membrane of acrosome-reacted sperm (Anderson et al., 1989).  Several  other monoclonal antibodies have been used to evaluate acrosomal status (Wolf et al., 1985; Moore et al., 1987). Currently there are no reliable markers for sperm capcitation in human sperm (Fichorova and Anderson, 1991). However, Mabs to surface antigens can potentially be used to recognize the changes of the sperm membrane associated with capacitation.  Other methods of monitoring capacitation and acrosome reaction include  chlortetracycline flourescence assay (Lee et al., 1987)  11  CHARACTERIZATION OF THE STX-10 ANTIGEN  A total of seventy-six monoclonal antibodies against sperm antigens were submitted to the World Health Organization (WHO) sperm antigen workshops in 1986 and 1989 (Anderson et al., .1987; Anderson, DJ, 1990). Out of these monoclonal antibodies, thirty-seven were reported to react to un-fixed human sperm. (The others were found to cross-react with somatic tissues). Using a combination of methods including carboxyl-fluorescein diacetate sperm viability, FITC-PSA (Pisum agglutinin-Fluorescein isothiocyanate), and rhodamine immunofluorescence assays, it was found that only five of the submitted antibodies genuinely reacted with surface antigens of viable un-fixed human sperm (Fichorova and Anderson, 1991). One of these five exclusive antibodies was S75, or HSA-10 Mab. In this study, the binding pattern of S75 showed the antigen to be localized in the acrosomal contents. The antigen was exposed after induction of the acrosome reaction by calcium ionophore A23 187. HSA-10 Mab was found to bind to the equatorial segment (Fichorova and Anderson, 1991). However, by immunogold localization and transmission electron microscopy, the STX- 10 antigen was found to be localized mainly on the inner acrosomal membrane of fixed sperm (Lee et al., 1993).  With the use of the Mab, HSA-10, we have been able to isolate and purify the STX-10 antigen and reveal its localization in sperm and placenta extract (Lee et al., 1993).  In  humans, it is found only in sperm and in the placentaltrophoblast. In mouse oocytes and  12  two-cell embryos, the STX-10 antigen which was localized on the cell surface, was studied by using a FITC-labelled HSA-10 Mab probe (Polgar et. al., 1994). They found that the mobility of the STX- 10 glycoprotein was greatly reduced in degenerated zygotes and twocell embryos in comparison to their viable counterparts. Glycoprotein mobility was even less in the membranes of unfertilized oocytes.  Upon cell degeneration, the STX-10  glycoprotein was either internalized or the HSA- 10 Mab was prevented from entering the cytoplasm by the plasma membrane.  With HSA-10 Mab as the probe, mouse oocyte  fertilization and pre-implantation embryonic viability can now be assessed (Polgar et al., 1994).  My research project involved the characterization of the human STX-10 antigen in two areas.  The first area concentrated on the confirmation of previous data obtained from  SDS-PAGE, Western blots, indirect immunofluorescent assays,  sandwich enzyme  immunoassays (SEIA) and enzyme-linked immunosorbent assays (ELISA).  This set of  experiments focused on the biochemical and immunological characterization of the STX- 10 antigen. The second area involved experiments in the molecular characterization of STX 10 antigen including screening of a human placenta cDNA library to obtain the gene. Following isolation and purification of cDNA clones expressing STX-10 related fusion protein, the cDNA inserts were analyzed by restriction enzyme digestions, PCR amplification, and Northern blots.  13  MATERIALS AND METHODS  CHEMICALS  All molecular weight standards used in protein chemistry and materials for immunoaffinity  chromatography, polyacrylamide and gel electrophoresis were supplied by BlO-RAD, Burlington, ON.  Materials for bacterial culture media were supplied by DIFCO,  Vancouver, BC. Disposable petri dishes of 90 mm and 150 mm, nickel ammonium sulfate, imidazole, and 8-well slides (for immunofluorescent assays) were purchased from Fisher Scientific Co., Ottawa, ON.  For immunofluorescent assays, ELISAs, SEIAs, Western  blots, and immunoscreenings, HRP and ALP-labelled goat-anti-rabbit/mouse-IgG-fA+M, and FITC-labelled goat-anti-rabbit/mouse-IgG+A+M, were all supplied by GIBCO BRL, Burlington, ON. GIBCO BRL also supplied Ham F-b  media, Fetal Calf Serum, and all  molecular biology supplies including restriction enzymes, and molecular weight standards. Polystyrene microtiter wells for ELISA and SEIA were supplied by Dynatech, Arlington, VA.  Calcium ionophore A23 187 and PSA-FITC, used in immunofluorescent assays;  diethyl pyrocarbonate (DEPC), guanidine thiocyanate, and sarcosyl, used for RNA isolation; and exposure cassette with intensiflying screen were supplied by SIGMA, St. Louis, MO. The 2gt1 1 immunoscreening kit was supplied by Clontech, Palo Alto, CA. Radioactive material, nitrocellulose filters, nylon membranes, and ECL detection reagents were purchased form Amersham, Oakville, ON. 2gt 11 sequencing primers were supplied by New England Biolabs, Mississauga, ON.  14  SOURCE OF ANTIBODIES  Mabs HSA-1O, HS-11, and MSA-63 were generated by the Hybridoma Core Facility of the Canadian Genetics Disease Network according to the published procedure (Lee et a!., 1984; Menge et al., 1987).  Rabbit anti-STX-1O sera and rabbit anti-MSA-63 sera were also produced by the Hybridoma Core Facility of the Canadian Genetics Disease Network according to standard procedure (Liu et a!., 1989).  SOURCES OF HUMAN SPERM AIJD PLACENTA EXTRACTS  Fresh human sperm samples were obtained from healthy sperm bank donors.  After a  suggested three day abstinence, samples were delivered by masturbation into sterile containers.  The samples were then processed as described in the methods.  Placenta  obtained from C-sections (Caesarean sections), was supplied by the Departments of Obstetrics and Gynaecology, from B.C. Women’s Hospital in Vancouver, British Columbia and from Veteran’s Memorial Hospital in Taipei, Taiwan.  15  PURIFICATION OF CHROMATOGRAPHY  THE  STX-10  ANTIGEN  BY  IMMUNOAFFINITY  An immunoaffinity column, composed of Affi-gel 10 with HSA- 10 Mab as the ligand, was used to purify the STX-10 antigen from human placenta extract and from human semen extract. According to the protocol supplied by BlO-RAD, HSA- 10 Mab (5 mg/mi in 0.1 M NaHCO , pH 8.3) was coupled to Affi-gel 10 at a density of 10-20 mg HSA-10 per ml 3 of Affi-gel 10. This was followed by incubation of the column with 1 M ethanolamine, pH 9.5 to block any uncoupled sites (Lee et. al., 1993). Frozen human placenta extract was  thawed, then centrifuged for 10 minutes at 10,000 rpm.  The supernatant was filtered  through cotton, the resulting volume was measured, and the absorbance at 280 nm was read. Human semen extract was homogenized, then centrifuged for 20 minutes at 10,000 rpm at 40C. Ammonium sulfate was added to the supernatant to precipitate any soluble proteins. The protein pellet was dissolved in phosphate-buffered saline (PBS) and dialyzed overnight in PBS at 4°C.  After centriftigation to remove any denatured proteins, the  semen extract was applied to the column. The 10 cc. immunoaffinity column was washed with 25 ml of 0.1 M giycine-HC1, pH 2.2. 50 ml of PBS was used to equilibrate the column before loading the column with either placenta or semen extract. The column was then washed with PBS+1 M NaC1 until the absorbance of the eluents were less than 0.01, when measured at O.D.280nm. The antigen was then eluted from the column using 0.1 M glycine-HC1 at pH 2.2. One milliliter fractions were collected into 15-20 separate tubes.  16  The fractions were immediately  neutralized with 1 M Tris-HC1, pH 8.0. The column was re-equilibrated with PBS. The protein concentrations of each fraction were estimated by spectrophotometery readings at an absorbance of 280 nm.  SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS  The peak fractions eluted from the HSA- 10 immunoaffinity column were concentrated in Centriprep Concentrator tubes from Amicon (Danvers, MA), and then examined by SDS PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) following Laemmli’s established method. The sample buffer was prepared with 10% SDS, bromophenol blue, J3-mercaptoethanol or DTT, iodoacetate, and sucrose. An equal volume of sample buffer was added to each eluted protein product. This mixture was boiled for 4 minutes and then loaded onto 10% polyacrylamide gels.  Electrophoresis was carried out in mini-gel  electrophoretic apparatus, at a constant voltage of 200 V. A low range molecular weight standard was used to estimate the size of the protein products.  The proteins in the  polyacrylamide gel were then either blotted onto nitrocellulose membrane or stained. To visualize the proteins, the gel was stained with either Coomassie Blue or Silver Stain.  17  WESTERN BLOT  The proteins on the 10% polyacrylamide gels were transferred to nitroceilulose membrane according to the established procedure of Towbin et. al. (Towbin, 1979). Transfer of the proteins took place overnight at 30 V in transfer buffer (25 mM Tris, 192 mlvi glycine, pH 8.3, 20%{v/v} methanol). The next morning, the nitrocellulose membrane was placed in 5% skim milk in PBS-0.5% Tween for 30-60 minutes at room temperature to block any unoccupied sites on the membrane.  After blocking, the strips prepared from the  nitrocellulose membrane were incubated in primary antibody solution (1:1000 dilution) for 3 hours at room temperature, on the shaker.  The primary antibody was either HSA- 10  Mab (1 mg/mi) or rabbit-anti-STX-10 Pab (1 mg/mI) with the corresponding controls: IgE44, and unrelated Mab or Normal Rabbit Serum. After primary antibody incubation, any unbound antibody was removed by washing three times with PBS-0.5% Tween. The strips were then incubated for three hours in HRP-labeiled secondary antibody, either goat anti-rabbit-HRP or goat-anti-mouse-HRP at 1:3000 dilution.  The HRP signal was then  developed by incubating the strips in a solution of 30 mg urea peroxide in 50 ml 0.1 M Tris-HC1, pH 8.0 plus 30 mg 4-chloro-1-napthoi in 10 ml methanol until a purple coloured band was visualized. The reaction was terminated by rinsing in 2 dH O .  18  ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)  Polystyrene microtiter wells were coated overnight at 4°C, with STX- 10 Ag purified from either placenta or semen extracts at 1-5 ug/mi in 50 mM Tris-HC1, pH 8.0. After removing the coating solution, non-specific binding sites were blocked for 20-60 minutes with blocking solution (PBS+l.0% BSA, pH 7.4). After aspiration of the blocking solution, the wells were air-dried.  STX- 10 Ag coated wells were secured in well holders. Primary antibodies, HSA- 10 Mab or rabbit-anti-STX-10 serum, and normal rabbit serum or IgE44 Mab controls were adjusted to 1 mg/mI. Serial dilutions were carried out from 1:100 to 1:3200 in PBS buffer containing 1.0% BSA and 0.1% TMS. Primary antibody was added (100 ul) to each well and incubated for 1 hour at 37°C. Excess antibody was removed by washing once with . The secondary antibody, HRP-labelled goat dH O PBS-0.5% Tween and three times with 2 anti-mouse-IgG+A+M or goat-anti-rabbit-IgG+A+M at 1:1000 dilution in conjugate buffer, was added and the wells incubated for 1 hour at 3 7°C. Unbound second antibody O. 2 was removed by washing three times with dH  The HRP label was developed by the  addition of 100 ul each of Solution A (0.57 g/L urea peroxide in 0.1 M citrate-phosphate, O, and 5% TIVIB in 2 pH 5.0) and Solution B (40% methanol, 10% glycerol, 45% dH DMSO).  The resulting blue colour product was developed for 15-20 minutes before  0 The colour intensity of the 4 S 2 H terminating the reaction with 100 uL of 2M HC1 or 2M .  19  micro-wells were then read at 450 nm on an automatic microplate reader.  In some of the  experiments, ALP-labelled second antibody was used instead of HRP-labelled antibody. In these cases, 100 ul of 1:3000 dilution of goat-anti-rabbit or mouse-IgG-I-A+M-ALP ) was 2 conjugate (2.6 mg/ml) in substrate buffer (0.1 M Iris, 0.05 M NaC1, 2mM MgC1 added to each well. The microtiter wells were read at 15 minutes, 1 hour, 2 hours, 3 hours and 6 hours for complete substrate development.  Absorbance readings were taken at  405 O.D.  SANDWICH ENZYME IMMJJNOAS SAY (SEIA)  HSA-10 Mab was coated on microtiter wells at 1 ug/ml in 50 m]VI Tris-HC1, pH 8.0. After blocking any non-specific sites, STX-10 Ag (1 mg/mI) from different sources was added in serial dilutions from 1:100 to 1:3200. STX-10 purified from human placenta and semen extracts were compared to each other and to crude extracts, for their reactivity to HSA-10. A one-step assay was carried out. After the addition of 100 ul antigen to each well, a sandwich assay was carried out by adding 100 ul of HSA-10-HRP conjugate (1:800 dilution) to the wells. The wells were covered and incubated at 37°C for three hours, then . dH O washed three times with PBS-0.5% Tween and three times with 2  The enzymatic  activity of the HRP-label on the wells was then determined by substrate development with Solution A and Solution B, as described under the methods for ELISA. Absorbance was read at 450 nm by a Molecular Device Microplate reader.  20  INDIRECT IMN4UNOFLUORESCENT ASSAY  Fresh human sperm samples were obtained from healthy sperm bank donors and allowed to liquefy at room temperature. Initial motility and concentration were determined. Each sample was then underlaid in Ham F- 10 medium for one hour in a 37°C incubator to allow swim-up to occur. The top layer containing motile sperm was collected, pooled, and then centrifuged for five minutes at 2000 x g. The pellet was resuspended with Ham F- 10 medium to the desired concentration of 106 spermlml. Post swim-up motility was noted and compared to initial observations.  HSA-10 Mab (1 mg!ml) was used to determine the location of the STX-10 Ag on human and mouse sperm.  Samples were either methanol-fixed onto 8-well slides before adding  primary antibody or samples were incubated with first antibody before fixing onto slide wells. Primary antibody dilutions ranged from 1:200 to 1:3200.  FITC-labelled secondary  antibody, goat-anti-mouse-IgG-+-A+M detected the monoclonal antibodies.  The sperm  samples were washed, then mounted before observation under both ultraviolet and visible light.  For time-dependent binding assays, human sperm samples were prepared as previously described. The post swim-up sample at 106 sperm per ml of Ham F-b  21  was kept at 37°C  for the duration of the experiment. At designated times (0 hr, 1 hr, 2 hr, 4 hr), 90 ul of sperm sample was removed and incubated with 10 ul of each primary antibody (1:100 dilution of 1 mg/mi) to be tested. Primary antibody: HSA-10, HS-11, rabbit-anti-STX-10 serum, or PSA-FITC was incubated with sperm for 30 minutes at 370C before coating on wells. Three or four wells were coated per antibody tested with 10 ul coated per well. Samples were allowed  to dry for 10 minutes on the 37°C dishwarmer plate before  methanol fixation. Each well was washed three times with blocking solution (PBS+0.5% BSA  +  0.1% TMS).  FITC-labelied secondary antibody, goat-anti-mouse or rabbit  IgG+A+M (at 1:1000 dilution) was added to all wells except PSA-FITC treated samples, and incubated for 30 minutes. After four hours, the acrosome reaction was induced by the addition of calcium ionophore A23 187 to sperm samples (final concentration lOuM). After different treatment times from 15-60 minutes, indirect immunofluorescent assay was performed on the samples as previously described. The percentage of positive staining at the designated times and the results of the acrosome reaction on binding affinity was observed. The percentage of positive staining was based on a total minimum count of 100 sperm from 10 different fields. There was a 10-20% error rate. Positive staining was determined to be strong fluorescence of the cap region, as opposed to faint fluorescence.  22  IMMUNOSCREENING OF HUMAN PLACENTA cDNA LIBRARY  Rabbit anti-STX-10 antisera were used to identify cDNA clones expressing STX-10 fusion protein from a human placenta cDNA library constructed in a gt1 1 vector. Instructions from Clontech’s immunoscreening kit were followed with the reported modifications. With 0 .2) as the host bacteria, the library was plated at a D. 0.1-0 . O. 60 = E. coli 1090r- (at 1 4 plaque forming units (pfu) per 90 mm petri dish. After three hours of density of 2 x i0 incubation at 42°C to induce lysogenic growth, IPTG saturated filters were placed on each plate. After a further three hour incubation at 3 7°C, the filters with transferred proteins were marked, removed from the plates, and treated with blocking solution (5% IGA Foods skim milk powder in PBS+0.5% Tween). Duplicate filters were made on each plate with one filter incubated in monoclonal HSA-10 antibody and the other filter in polyclonal anti STX- 10 serum, at room temperature, overnight on the shaker. The filters were incubated for three hours with HRP-labelled second antibody and followed with peroxidase substrate development (400 mg imidazole, 800 mg fickle ammonium sulfate, 15 mg diaminobenzidine ,and 60 ul of 30% urea peroxide in 150 ml of 50 mlvi Tris-HC1, pH 7.6). Between primary and secondary antibody incubations, the filters were washed three times in TBS-Tween or PBS-Tween, for 10 minutes per wash.  Between the second antibody incubation and  developments, filters were washed three times in TBS or PBS. Normal rabbit serum and no primary antibody incubation were used as the negative controls.  Positive control,  chicken ovalbumin ?gt 11 with goat-anti-chicken ovalbumin primary antibody was supplied  23  by Clontech (Palo Alto, CA). Positive clones, seen as dark purple rings on the filters, were picked from the original agar plates. Each clone was eluted overnight at 4°C in Lambda Dilution Buffer (LDB) (5.8 g , 50 ml 1M Tris-HCI, pH 7.5, and 0.01% gelatin in one liter 7H 2 4 MgSO NaCl, 2.0 g 0 , pH 7.5) with 10% chloroform. dH O 2  Each clone was titered, and purified through  additional rounds of screening until all the phage clones showed 100% positive staining to anti-STX-10 sera.  ISOLATION AND PURIFICATION OF cDNA CLONES  The cDNA inserts were isolated from clones according to Promegas instructions described here with some minor changes.  Transfected 1O9Or- bacteria cells were mixed with  melted soft top agarose and poured onto LB agarose plates. After the agarose had cooled (10-15 minutes), the plates were incubated at 37°C for 6-7 hours until the plaques almost lysed the entire surface. 12 ml of LDB was added to each plate. After overnight elution at 4°C, the buffer was removed and centrifuged to pellet the bacterial debris,  The  supernatant was then transferred to a 50 ml Sorvall centrifuge tube to isolate the bacteriophage DNA.  RNase A and DNase I (RNase-free) were added to the supernatants to final concentrations of 1 ug/mi and were incubated at 37°C for 30 minutes. An equal volume of 20% PEG/2 M  24  NaC1 solution was added and incubated for 1 hour at 0°C.  PEG absorbed to the  bacteriophage and the high salt concentration (2 M) was necessary to form a nucleic acid precipitate.  The tubes were centrifuged at 10,000 rpm for 20 minutes to recover the  precipitated phage particles. Supernatants were removed and the pellets resuspended with lx LDB. After a 2 minute centrifugation to remove debris, supernatants were transferred to new 1.5 ml microcentrifuge tubes. 5 ul each of 10% SDS and 0.5 M EDTA, pH 8.0 (to inhibit DNase) were added and incubated at 68°C for 15 minutes. The solution was then extracted twice with an equal volume of chloroform only. protein and contaminants from the samples.  These extractions removed  Buffer-saturated phenol (GIBCO BRL,  Burlington, ON) functioned in denaturing proteins and inhibiting enzymatic function while With each extraction, the mixtures were  chloroform functioned in extracting lipids.  vortexed for 30 seconds before centrifugation at 12,000 rpm at 4°C for 5 minutes. The upper aqueous phases (containing nucleic acid) were then transferred to new tubes for the next extraction.  To the last extraction, an equal volume of isopropanol was added to  precipitate the sample. After mixing, the solution was left at -20°C for 20 minutes. The tubes were then centrifuged at 14,000 rpm for 10 minutes. The pellets were vacuum-dried, then washed with 70% EtOH. Finally, the pellets were resuspended with TE buffer (Tris EDTA, pH8.0).  After quantitation of the DNA isolated from each clone, the DNA samples were then digested with EcoRI or double-digested with KpnI/SstI at 10 units per ul of DNA,  25  overnight at 370C.  The products were then examined on a 1.0 or 1.2% agarose gels  depending on the expected fragment sizes.  PCR AMPLIFICATION OF cDNA  The polymerase chain reaction was carried out according to standard procedure (Sambrook et al., 1989).  The 2gt11 forward and reverse primers were employed for  amplifying the DNA isolated from the clones.  The 24 mer sequence of each primer is  given: forward primer-d(GGTGGCGACGACTCCTGGAGCCCG) and reverse primer d(TTGACACCAG-ACCAACTGGTAATG). PCR reactions were carried out in lOx Taq buffer, 50 mM MgCI , 5 mM dNTP’s. The PCR conditions were as stated: 30 cycles of 2 95°C for 1 minute, 67°C for 2 minutes, and 72°C for 1 minute. A 10 minute extension at 720C was added.  The resulting PCR products were separated on 1.2 % agarose gels  containing ethidium bromide.  AGAROSE GEL ELECTROPHORESIS  According to standard procedure (Sambrook et al., 1989), 1.0 or 1.2% agarose gels were prepared from ultrapure DNA agarose in 50 mlix TAE buffer (0.04 M Tris-acetate, 0.001  26  M EDTA, pH 8.0) and 0.5 ug/ml Ethidium bromide.  lOx sample buffer (50% glycerol,  0.05% bromophenol blue, 0.05% xylene cyanole, lx TAE buffer) was added to each  sample before loading onto gel slots. The gel was run in lx TAE buffer at a voltage of 2-3 V/cm for 1.5 hours, with a 1 kb standard ladder supplied by GIBCO BRL.  WESTERN BLOT OF FUSION PROTEINS  Plate lysates of the 1 kb clone (8B2, 8E1) were prepared to detect the size of the fusion protein produced in this clone. Non-transfected E. coli and clone MSA-63 were used as controls.  Plate lysates from clones 8B2, 8E1, and MSA-63 were prepared under the following conditions. In order to obtain a titer of 1 pfu/ml, 200 ul of each clone at 1 0 dilution, was used to transfect 200 ul of Y1090r bacteria cells (at 650 O.D. nm= 1.4). These cells were then mixed with 7 ml of melted soft top agarose and poured onto LB plates. After the agarose had cooled to room temperature, the plates were incubated at 370C until the plaques almost lysed the entire surface. The agarose surface was scraped into a 50 ml Sorvall centrifuge tube. An equal volume (7 ml) of SDS sample buffer (4.28 g sucrose, 3.75 g SDS, 0.95 g Tris, 0.093 g Na EDTA, 22.5 ml dH 2 O, adjusted to pH 6.9), was 2 added. The tubes were allowed to sit overnight at 40C. The next morning the samples  27  were heated at 37°C for 15 minutes.  Next, the samples were homogenized, then  centrifuged at 10,000 rpm, for 15 minutes and then the supernatants were transferred into new tubes.  In eppendorf tubes, 5 mg of DTT and 5 ul of 0.05% bromophenol blue were added to 100 ul of the each sample. The samples were then boiled for 2 minutes and applied onto 8% polyacrylamide gels. detection system.  Western Blot assay was carried out accompanied with an ECL The membranes were rinsed in TBS before incubation in 5% milk  blocking solution. The membranes were incubated in rabbit anti-STX- 10 (1 mg/mi), rabbit anti-MSA-63 (1 mg/ml), or normal rabbit serum (1 mg/ml) at 1:1000 dilution..  After  washing, and further incubation with HRP-labelled goat-anti-rabbit secondary antibody (1:2000 dilution), the strips were incubated for  1  minute in ECL (Enhanced  chemiluminescence) detection reagent. The strips were then drained of excess reagent and wrapped in plastic wrap. In the darkroom, Hybond-ECL film (Amersham, Oakville, ON) was exposed to the membranes for 1 mm, 3 mm, 5 mm or 10 minutes, and then processed for the HRP signal.  28  DNA SEQUENCING  The 1 kb PCR product was given to the Canadian Genetics Diseases Network Facility for DNA sequencing  .  The “Taq DyeDeoxy terminator Cycle Sequencing Kit” from Applied  Biosystems, was used for sequence analysis.  The protocol used at the Facility will be  described here briefly.  O, dNTP’s, buffer, 2 The template (PCR product), gt1 1 forward and reverse primers, dH DyeDeoxy Terminators and AmpliTaq DNA Polymerase were mixed together. The mix was cycled 25 times through a Perkin Elmer 480 thermal cycler, under the following conditions: 96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 4 minutes.  The  extension products were purified by passing the samples through spin columns to remove excess dyedeoxy terminators. The samples were then mixed with foramide and EDTA, and denatured by heating at 90°C for 2 minutes before loading onto the DNA sequencer. Each dideoxynucleotide was labelled with a different fluorescent dye and these labels were incorporated into the DNA. As the sample in the gel passed through, the laser beam at the bottom of the gel picked up the fluorescent label and sent it to the computer program.  Four primers were generated by the UBC oligonucleotide synthesis lab based on the newly derived DNA sequence.  29  ISOLATION OF RNA  Total RNA was isolated from BeWo human choriocaricnoma cells, purchased from ATCC (American Type Culture Collection, Rockville, MD), and cultured in our laboratory. Single step RNA isolation was carried out based on the established method (Chomczynski and Sacchi, 1987).  BeWo cells were washed and resuspended in Solution D (2-3-  mercaptoethanol, guanidine thiocyanate, diethylpyrocarbonate treated water, 0.75 M sodium citrate, and 10% Sarcosyl). Resuspended cells were mixed with 2 M NaAcetate, pH 4.0, phenol, and chloroform:IAA.  After centrifugation at 10,000 rpm for 15-20  minutes at 40C, the aqueous layer was transferred to a new tube. Extraction with phenol and chloroform:IAA was repeated. The aqueous layer was mixed with isopropanol for 1 hour at 2O0C, then centrifuged at 10,000 rpm for 15-20 minutes at 4°C. The pellet was resuspended in 300 ul of Solution D and then precipitated with an equal volume of isopropanol at -20°C. Following centrifugation at 4°C for 10 minutes at 10,000 rpm, the pellet was resuspended in 75% ethanol and vacuum-dried,  Finally, the pellet was  resuspended in 20 ul of 2 DEPC-dH and RNA concentration was measured in the O spectrophotometer.  30  NORTHERI BLOT  The isolated RNA was transferred from a 1 1% formaldehyde gel to nylon membrane following the method of Lehrach, 1977.  10 ug of RNA was mixed with 25 ul of RNA  sample loading buffer (380 ul deionized foramide, 75 ul lOx MOPS, 120 ul formaldehyde, 63 ul 80% glycerol, 40 ul 10% bromophenol blue, and 33 ul 1 mg/mi ethidium bromide). The samples were heated at 65°C for 15 minutes, loaded on a 1.1% formaldehyde minigel, and separated at 80 V.  The gel was soaked in lOx SSC before the RNA was  transferred by capillary action, overnight.  The RNA was cross-linked to the nylon  membrane by baking at 80°C for 2 hours. In collaboration with Biao-Yang Lin from Dr. Michael Hayden’s laboratory, the blot was then probed with the 32 P-labelled 1 kb cDNA fragment.  31  RESULTS  PURIFICATION ANL) CHARACTERIZATION OF THE STX-10 ANTIGEN  Using HSA-10 Mab as the ligand, STX-10 antigen was purified from human placenta by immunoaffinity chromatography. An elution profile of a typical purification of STX- 10 from 20 ml of placenta extract is shown in Figure 1. In this case, the peak fractions (from 0.018 to 0.034 at O.D.280 nm) were eluted in tubes #3-#6. These protein fractions were concentrated and examined on 10% polyacrylamide gel. From placenta extract, STX-10 was confirmed to be a protein of 75±5 kD.  In some cases, a single major band was  observed and in others, an aggregate of three bands.  From semen extract, the same  band(s) were observed but at a lower concentration. In addition to this, there were major bands observed between 10-20 kD. These results from SDS-PAGE are shown in Figure 2.  The purified STX-10 antigen was transferred to nitrocellulose and analyzed by Western blot assay to determine the molecular size of the STX-10 protein. Rabbit-anti-STX-10 serum recognized only the 75 kD band from placenta and semen extracts as shown in Figure 2. The lower molecular weight bands from sperm extract were not immunoreactive. The 75 kD protein was not recognized by HSA-10 Mab (1 mglml at 1:500 or 1:1000 dilution) or the negative controls, IgE44 Mab and normal rabbit serum.  32  Figure 1.  Elution profile of the STX-1O antigen purified by an HSA-1O immunoaffinity column. The absorbance of each fraction at 280 nm was plotted against tube numbers. This graph shows a typical profile of STX-10 antigen isolated from 20 ml of placenta extract. Tube numbers are as indicated.  33  S1qw,itj qn OL 00.0  00 >  C 0  &  ,’ 7r ‘J\J  (  cc  £00  Figure 2.  Sodium dodecylsulfate polyacrylamide gel electrophoresis (10% gels) and Western blots of purified STX-10 antigen. In Lane 1, 75 kD STX-10 Ag purified from placenta extract; in Lane 2, the corresponding Western blot is shown. In Lane 3, molecular weight standards, from top to bottom, are: Phosphorylase B, 106 kD; Bovine serum albumin, 80 kD; Ovalbumin, 49.5 kD; Carbonic anhydrase, 32.5 kD; Soybean trypsin inhibitor, 27.5 kD; Lysozyme, 18.5 kD. In Lane 4, the STX-10 Ag purified from sperm extract is shown; in Lane 5, the corresponding Western Blot. In Lane 6, the negative control is shown. {Photo from Lee et al., 1993}  35  7’ fl/ F.  CA)  L  •1-.•  ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)  ELISA was employed to study the binding affinity and specificity between relevant antibodies (HSA-10 Mab, rabbit-anti-STX-10 polyclonal antibody) and the STX-10 antigen coated on microtiter wells. Similar immunoreactivities were observed for antibody binding to semen extract STX- 10 and placenta extract STX- 10. At initial concentrations of 1 mg/ml for HSA-10 and undiluted serum for anti-STX-10, antigen-antibody binding was found (a) to be dose-dependent, and (b) to be significantly higher with polyclonal anti STX-10 sera.  SANDWICH ENZYME IMMUNOAS SAY (SEIA)  In view of the fact that the native form of STX- 10 protein has multiple epitopes to HSA 10, a solid phase sandwich enzyme immunoassay was performed.  To determine the  immunoactivity of the STX-10 antigen, the antigen was sandwiched between HSA-10 Mab coated on wells and HRP-Iabelled HSA-10. As seen in Figure 3, dose dependent curves were obtained at absorbance 450 nm. Similar results in immunoactivity were observed for the STX- 10 antigen purified from placenta and semen extracts.  37  Figure 3.  Sandwich enzyme immunoassay with HSA- 10 Mab as the coated antibody on wells and as the detecting antibody labelled to HRP. The different reactivies of soluble placenta extract  (—&-), crude semen extract (•),  purified STX-10 from human placenta (-0-), seminal plasma negative control  (-s), and  (-.-) at serial dilutions were read at an absorbance of  450 nm. Initial protein concentrations of each homogenate were  determined by absorbance at 280 nm. Approximate protein concentrations are as follows: 20-25 mg/mi placenta extract, 45-55 mg/ml semen extract, 45-55 mg/ml seminal plasma, and 1 mg/ml STX-10 antigen.  38  00001. •  0001.  iopj uoiInpQ  001  I.  01. 1. ‘0  • P.O • 90 • 90 •01.  STX-10 OF LOCALIZATION IMMUNOFLOURESCENT ASSAY  ANTIGEN  IN  HUMAN  SPERM  BY  By using indirect immunofluorescent assay, it was confirmed that HSA- 10 Mab reacted specifically to the acrosomal region of methanol-fixed human sperm (Lee  Ct  al., 1993).  This was observed under ultraviolet light as shown in Figure 4. Under the same conditions, rabbit-anti-STX-10 was found to also react to the same acrosomal region. The negative control, a non-related Mab, did not show fluorescence. Both HSA-10 Mab and rabbit-anti STX-10 did not bind to mouse sperm.  Serial dilutions of the primary antibody (1 mg/ml) from 1:200 to 1:3200 were compared to determine the optimal dilution for visualization. primary antibody was determined to be 1:400  -  For HSA-10, the optimal dilution of  1:800 dilution.  An indirect immunofluorescent assay of human sperm was carried out to observe the timedependent changes of HSA- 10 binding to human sperm under different physiological conditions. These results are shown in Figure 5. From three different sperm donors, it was generally observed that the percentage of HSA-10 binding to sperm increased slightly from 0 hours to 4 hours.  After the induction of the acrosome reaction by 10 uM A23 187  calcium ionophore for 15 minutes, the binding of HSA-10 Mab to human sperm significantly increased (100 sperm counted, 10-20% error). In this study, PSA-FITC was  40  used an indicator of uncapacitated and capacitated sperm. It is known that PSA does not bind to acrosome-reacted sperm (Cross, 1986).  41  Figure 4.  Indirect immunofluorescence localization of STX-10 antigen on methanol fixed human sperm. HSA-10 Mab (1:1000 dilution of lmg/ml) was used as the primary antibody. A: Acrosomal cap fluorescence observed under UV light. B: Same field observed under visible light.  42  Figure 5.  Time-dependent changes of HSA-O Mab binding to human sperm as detected by indirect immunofluorescent assay. Time-dependent changes from three different donors were observed at 0 hours, 2 hours, 4 hours, and after induced acrosome reaction. The three donors are indicated as follows: JRK  (—ri-),  JKH (-e-), and LAL (-a-). A graph of the proportion of sperm  exhibiting fluorescence is shown. FITC-Iabelled PSA served as the positive control in each case.  44  U,  20  40  60  80  100  Ohr  41w  TIME COURSE  2hr  4 hr + induced acrosome reaction  IMIVIUNOSCREEMNG OF HUMAN PLACENTA eDNA LIBRARY  Several trials of immuno screening a human placenta eDNA library were carried out. In the first trial, the immunoscreening of 2 x io5 clones resulted in rabbit-anti-STX-1O serum hybridizing to five clones.  These clones were sequenced at the University of North  Carolina, in Chapel Hill, NC. The results obtained were unexpected. Of the five clones, only three were able to be sequenced. Two were found to be identical and matched the database sequence for phosphatidylserine synthetase.  However, this was the incorrect  reading frame for the 3-galactosidase protein. The other clone was found to match the database sequence for filamin, an actin-binding protein. Due to the false-positive results, a different batch of rabbit-anti-STX- 10 serum was produced and another trial of screening was carried out.  The second trial of the immunoscreening of 4 x i5 clones resulted with only one independently derived clone that was recognized by rabbit-anti-STX-10 serum.  Clones  8B 1, 8B2, 8D 1, 8D2, 8E 1, and 8E2 were found to be identical by PCR amplifications and by restriction enzyme digestions with KpnI/Sst I.  The third trial of immunoscreening  resulted with six positive clones: 1-1, 2-1, 2-2, 7, 8, and 9 from a primary screening of 400,000 clones. These seven clones were transferred to duplicate filters and screened with either rabbit-anti-STX-10 serum or HSA-10 Mab. While polyclonal antiserum was able to detect positive clones, monoclonal antibody was unable to detect recognize any positive  46  clones. The cDNA isolated from positive clones during the third trial of immunoscreening were double-digested with restriction enzymes Kpn I/Sst I. No cDNA insert was observed in these clones.  WESTERN BLOT OF PLATE LYSATES OBTMNED FROM cDNA CLONES  The fusion protein produced within the plate lysates was used to prepare Western blots. Plate lysates from clones 8B2, 8E1, and MSA-63 were transferred onto nitrocellulose and probed with various antibodies. Non-transfected E. co/i was used as the negative control. In rabbit anti-STX- 10 serum, an extra band at 140 kD was observed only from clones 8B2 and 8E1 (both 1 kb).  This band is indicated in Figure 6.  Many common bands were  observed between 8B2, 8E 1, MSA-63 and E. co/i. The 8B2 and 8E 1 strips incubated in normal rabbit serum did not show the extra 140 kD band.  47  Figure 6.  Western blot of fusion protein expressed by I kb clone. In lane 1, homogenate of E. coil only in soft top agarose, is shown. In lane 2, homogenate of top agarose containing E.coii lysed by clone 8B2, is shown. Molecular weight standards from top to bottom: Myosin, 205 lcD; 3-galactosidase, 116.5 kD; and Phosphorylase B, 77 kD. (This work was carried out with the assistance of Dr. Tatsuhiro Yoshiki.)  48  N  0  ANALYSIS OF DNA ISOLATED FROM POSITIVE CLONES  The plate lysate and liquid culture methods were used to isolate the bacteriophage and extract cDNA from the positive clones. Although foreign cDNA fragments are inserted in the unique EcoRl site of gt1 1 bacteriophage, only one clone was digested with EcoRI restriction enzyme. This unique site may have been lost or modified in our clones, during library cloning, since the positive controls (chicken ovalbumin cDNA and MSA-63 cDNA) were readily cut by EcoRI.  Using a double digestion with KpnI and SstI restriction  enzymes, the clones were found to (a) contain no cDNA insert (clones 1-1, 2-1, 7, 9), or (b) contain a 1.1 kb fragment (clones 8B1, 8B2, 8D1, 8D2, 8E1, 8E2). Other clones (8, 8C2) were not digested by KpnI/SstI.  Polymerase chain reaction (PCR) was employed to confirm the cDNA insert size of each clone and to amplify the product. This reaction was carried out using gt 11 forward and reverse primers based on the DNA sequence of the f3-galactosidase/EcoRI site.  cDNA  insert sizes varied from 88 bp to 3000 bp. PCR confirmed the data obtained by restriction enzyme digestions.  The 1 kb PCR product was sequenced at the Canadian Genetics Disease Network DNA Sequencing Facility. Four new primers specific to 8B2, termed GL#5, GL#6, GL#7, and  50  GL#8, were constructed based on the newly obtained DNA sequence.  These primer  sequences are listed in Appendix A. In Figure 7, the six possible reading frames for the 1 kb are shown. The resulting consensus DNA sequence and the location of the primers are shown in Figure 8. Also shown in Figure 8 is the unique EcoRI site, the 3-galactosidase gene, and the derived amino acid sequence. The cDNA insert was 1050 bp in length and contained a portion of the open reading frame of either 108 (frame 1) or 513 (frame 2) base pairs. An mRNA consensus degradation sequence (ATTTA) was found in the 3’untranslated region.  Homology searches of the resulting nucleotide and amino acid  sequences were carried out in the GENINFO(R) BLAST Network Service. In Appendix B, a summary of the results of the searches are shown. The search with the STX-10 cDNA 1 kb sequence revealed a segment that had 100% identity to 411 bp of a 449 bp cDNA fragment, clone s394 from human HepG2 3-directed MboI cDNA of human male liver (Okubo et al., 1992). However, this did not overlap with the open reading frame of the STX-10 gene. Instead, the sequences were homologous in the non-coding region from nucleotides 563 to 973.  51  Figure 7.  Possible open reading frames for 1 kb cDNA sequence. The different reading frames are as indicated. Arrows represent directions from 5’ to 3’, for frames 1-3 and for frames 4-6.  52  c..J  LI.  6  5  4  3  2  1  FRAME  READING  .  100  —  I  4,  200  200  -U  100  5’ to 3’ 400  300  400  —  300  500  500  600  800  700  800  —  700  -  600  3’ to 5$  900  900  Figure 8.  Nucleotide sequence of the placenta STX-1O 1 kb cDNA clone and deduced amino acid sequence. A. Open reading frame 1. B. Open reading frame 2. The number to the right indicates the positions of the nucleotides. The f3-galactosidase gene is indicated by  ****•  The mRNA degradation  signal (ATTTA) is underlined. The positions of the four newly generated primers (GL#5,6,7,8) are shown.Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Tie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; W, Trp; Y, Tyr.  54  Q, Gln; R, Arg; S, Ser; T, Thr; V, Val;  A  CGACTCCTGGAGCCCGTCAGTATCGGCGGAATVC GGG CAT GCA OTA TGG AAA CAA K A V ‘V Q G H AAC AAT CTC CAC CAT GAA AGT GAT GCA GTT TCA GGG TAT GAA ACG CAA AGC S LH H SG YET Q NN ES DAV  106  CAG TTC TCC COT CCC CCT ACC TCC AGT CAC ACA CCT TG.4 CCTCACCCCCAGCCC  160  Q F  S  R  P  P  T  S  S  H  T  P  STOP  TGATGTGCCTCTCACCATCATGAAAAGGAAACTGATGAACACCAATGATCTGGAGG AGTCCAGGCAGCTCACGGAGGAGATCCAGCGGCATCTGGATGCCAGGCACCTCATTG AGAAGTCAGTGCGTAAGATCGTCTCCTTGCTGGCAGCGTCCGAGGCTGAGGTGGAGCA  216 273 331  GL#5 GCTCCTGTCCGAGAGAGCCCCGCTCACGGGGCACAGCTGCTACCCAGAGGCCCTGCTG CACTTCCGGACCCACTGCTTCAACTGGCACTCCCCCACGTACGAGTATGCGTTGAGAC GL#7 ATTTGTACGTGCTGGTCAACCTTTGTGAGAAGCCGTATCCACTTCACAGGATAAAAT  503  TGTCCATGGACCACGTGTGCCTTGGTCACTACTGAAGAGCTGCCTCCTGGAAGCTTTT CCAAGTGTGAGCGCCCCACCGACTGTGTGCTGATCAGAGACTGGAGAGGTGGAGTGA GAAGTCTCCGCTGCTCGGGCCCTCCTGGGGAGCCCCCGCTCCAGGGCTCGCTCCAGGAC  617 675  CTTCTTCACAAGATGACTTGCTCGCTGTTACCTGCTTCCCCAGTCTTTTCTGAAAAAC GL#8 TACAAATTAGGGTGGGAAAAGCTCTGTATTGAGAAGGGTCATATTTGCTTTCTAGGA GGTTTGTTGTTTTGCCTGTTAGTTTTGAGGAGCAGGAAGCTCATGGGGGCTTCTGTA GL#6  853  GCCCCTCTCAAAAGGAGTCTTTATTCTGAGAATFTGAAGCTGAAACCTCTTTAAAT  910  CrFCAGAATGATTTTATTGAAGAGGGCCGCAAGCCCCAAATGGAAAACTGTTTTT  966  AGAAAATATGATGATTTTTGATTGC 11 11 GTATTTAATTCTGCCCC  1024  GAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCT  1050  B.  CGACTCCTGGAGCCCGTCAGTATCGGccTTCG GGC ATG CAG TATGGA AAC AP ACA ATC TCC ACC  S  G M  Q. Y G  N K  T  I  S  68  T  ATG AAA GTG ATG CAG 1TF CAG GGT ATG ftiAA CGC AAA GCC AGTTCT CCC GTC CCC CTA CCTCCAGTC  132  MKVMQFQGMKRKASSPVPLPPV ACA CAC CiT GAC CTC ACC CCC AGC CCT GAT Git CCT CTC ACC Alt ATG AAA AGG AAA CTG ATG AAC  THLDLTPSPDVPLTIMKRK  ACC AAT GAT CTG GAG GAG TCC AGG CAG CTC ACG GAG GAG Alt CAG CGG CAT CTG GAT GCC AGG CAC  TNDLEESRQLTEE  IQRHLDARH  TCATF GAG AAG TCAGTG CGTAAG Alt GTCTCC’ITG Cit GAGCG TCC GAG GCT GAG GTG GAG CAG  LIEKSVRKIVSLLAASEAEVEQ Cit Cit TCC GAG AGA GCC CCG CTC ACG GGG CAC AGC TGCTAC CCA GAG GCC CTG CTG CAC 1TC CGG  LLSERAPLTGHSCYPEALLHFR ACC CAC TGC 1TC AAC TOG CAC TCC CCC ACG TAC GAG TATGCG TI’G AGA CAT TTG TAC GTG CTG GTC  THCRNWHSPTYEYALRHLYVLV AAC CiT itT GAG AAG CCG TAT CA CiT CAC AGG ATAAAATTG TCC ATG GAC CAC GTG TGC Cr1’ GGT  KLSMDHVCLG  NLCEKPYPLHRI  CA C TA C TG A AGAGcTGCCrCC1tGiVGcF1TfCCMG1OitAGGcccCcCGAcTGitTOC1tAltAGAGAC H  y  198  LMN  STOP  264 330 396 462 528 600  1GGAGAGGTGAGTGAGAAGitGCitCitGGGcGcItCitGGGAGcGccGc1tCAGGGC1tGCitAGGAwT  677  1tyr\cAGA1tACuGcJtGCTGyrACCmCTTtCcCAGiunTurGAwCTACAAAlTAGGGTGGGAAki GL$  753  GcUIOTAT1tAGMGGGTCATAYITGCITFCTAGGAGGTfltTIO1TI1OCC1UJTAGTfl]tAGGAGCAGGAAGC GL#6  830  TCA1tGGGGCfltTGTAGCCCCitltAGGAG1ULTFA1]UtAGAAT11OMGCitAk?\CC1UflTAMTCrrC  907  AGA4TGAFI1 IA1TGAAGAGGGCCGCAAGCCCCAAATGGAAAACTG1TEl TAGAAATATGATOATI 11 IGATIt  989  C]TGTAflTMYtCCCCGA&YrnCAGCrGAGCGCCGG1OGCfACCAUACCAGTIOGTCF ***** **************************************************  56  l05 c  Figure 9.  Kyte-Doolittle hydropathy plot of the deduced amino acid sequence. A. Based on open reading frame 1 (36 amino acids). B. Based on open reading frame 2 (168 amino acids). The number of amino acids is indicated by the numbers below each plot. {Courtesy of Biao-Yang Lin}  57  B  c,.  A  =  -V  0 0  I  .—  C-)  .4—  Z7’  .v  0 0  •8  C)  -I  5.00 4.00 3.00 2.00 1.00 0.00 —1.00 —2.00 —3.00 —4.00 —5.00  5.00 4.00 3.00 2.00 1.00 0.00 —1.00 —2.00 —3.00 —4.00 —5.00  =  7 Scale =  Kyte-Doolittle  20  =  7  40  60  Scale  =  Kyte-Doolittle  80  100  120  140  160  __ __  __  __  Hydrophilicity Window Size  5 10 15 20 25 30 35 FGHAVWKQNNLHHESDAVSGYETQSQFSRPPTSSHTP*  Hydrophilicity Window Size  NORTHERN BLOT ANALYSIS  The BeWo choriocarcinoma cell line is recognized by the HSA-1O Mab and is known to express a high concentration of STX-1O antigen on the cell surface (WHO sperm antigen workshop III; Yang et al., in press). Since these cells were recognized by HSA-1O Mab, RNA was isolated from this cell line for Northern Blot assay.  The isolated RNA was  separated on a 1.1% formaldehyde-agarose gel. The 1 kb fragment was labelled with P-32 and used to probe the gel. As seen in Figure 10, the resulting mRNA (1.91 ug/ul) was found to be 5.5 kb in size. From mouse testes (2.06 ug/ul), a strong band was observed at 3.5 kb and a weaker band at 5.5 kb was also detected.  59  Figure 10.  Northern Blot of BeWo cells and mouse testes probed with radioactivelylabelled 1 kb cDNA fragment. On the left hand side, the corresponding 1%-agarose-formaldehyde gel is shown with the mRNA isolated from mouse testis (2.06 ug/ul) (lane A) and BeWo cells (1.91 ug/ul) (lane B). The molecular weight markers from top to bottom are as follows: 9.49 kb 7.46 kb 4.40 kb 2.37 kb 1.35 kb  0.24 kb. The Northern Blot is shown on the right hand side. In BeWo chorio carcinoma cells, mRNA was found to be 5.5 kb in size (lane B). In mouse testes, mRNA was found at 3.5 kb with a weak signal at 5.5 kb (lane A).  These blots were probed by Biao Yang Lin from Dr. Michael Haydens lab, Department of Medical Genetics.  60  w  w  cJ1  61  .  ,4.)  0 Cl)  Cl)  DISCUSSION  The STX-10 antigen, a 75 kD protein,  was confirmed to be present in both human  placenta and human sperm. The lower molecular weight bands (less than 2OkD) found only in sperm extract were not recognized by rabbit-anti-STX-10 serum.  These low  molecular weight bands may either have been co-purified with the STX- 10 antigen due to non-specific association, or may be degradation products due to sperm proteases such as acrosin and sperminogen.  Kamada et al. (1991) found that small molecular weight  proteins of 16-20 kD bound to IgG and the Fc fragment of antibodies. Since HSA-10 is in the IgG subclass, it is likely that the low molecular weight bands observed in our studies were those that bound to IgG.  In both Western blots and immunoscreening, HSA- 10 Mab was unable to recognize the STX-10 protein transferred onto nitrocellulose. Rabbit-anti-STX-10 serum was used to recognize the transferred protein and showed strong binding; meanwhile, the specific epitope recognized by HSA-10 was likely denatured or masked during SDS-PAGE or during transfer to nitrocellulose. Polyclonal antisera are generally recommended as probes for immunoscreening since monoclonal antibodies recognize only a single epitope (Huynh et al., 1985). Lee et al. (1993) suggested that the specific epitope of STX-10 was a peptide epitope which may be conformational in nature.  62  All our rabbits were immunized with STX-10 purified from human placenta and not human semen extract. Nevertheless, this rabbit antisera was capable of recognizing the same protein on the sperm acrosome. This observation provides strong evidence of the specificity of the anti-STX-10 sera to human sperm antigen. Ideally, polyclonal antiserum against semen extract  STX-10 should be produced and compared for differences in  immunoreactivity in the immunofluorescent assays, ELISA, SEIA, Western blots and immunoscreening experiments. However, it is expected that the results would be similiar since STX-10 is a specific epitope.  The ELISA assay was used to determine the specificity and the antibody titer of immunized animals.  Using SEJA, the immunoactivity of STX-10 was quantitatively  determined. The immunoactivity of STX-10 in different tissues was also tested using this assay. Previous data found that only human placenta and sperm extracts showed STX- 10 immunoactivity (Lee et al., 1993).  Further support for the specificity of the STX-10  antigen in gamete tissue comes from the absence of staining in immunocytochemistry studies with human heart, brain, liver, kidney, pancreas and blood cells (Yang et al., in press).  With the indirect immunofluorescent assay, binding of HSA- 10 Mab to human sperm was found to increase after the acrosome reaction was induced by calcium ionophore A23 187. This increase in binding was due to the exposure of STX-10 antigen, which is localized on  63  the inner acrosomal surface. In normal sperm, Schill et al. (1988b) found that the number of acrosome-reacted sperm increased spontanteously over a time course of 6 hours. The sperm population is asynchronous, and therefore undergoes capacitation and the ensuing acrosome reaction at different times. PSA was used as the positive control to indicate the presence of acrosome-intact sperm since PSA does not bind to acrosome-reacted sperm (Cross et a!., 1986; Cross and Meizel, 1989). PSA is a lectin which binds to specific sugar groups of the intra-acrosomal content or the outer acrosomal membrane (Cross and Meizel, 1989). After the membrane changes associated with the acrosome reaction, PSA is no longer capable of binding due to the loss of acrosomal content and outer acrosomal membrane. FITC-PSA and antibodies are considered good markers of sperm due to the strong intensity of labelling of acrosome-intact sperm and therefore, the strong differentiation between acrosomal and non-acrosomal materials (Cross and Meizel, 1989).  It was confirmed that HSA-10 Mab did not react with mouse sperm by indirect immunofluorescent assay.  It has been reported that this antibody does not cross-react  with mouse, hamster, chimpanzee and baboon sperm, testis, or epididymis (WHO sperm antigen workshop III report, 1992). It is interesting that STX- 10 is present in human sperm and placenta/trophoblast and mouse oocyte/embryo but not detected in mouse sperm. However, the Northern blot of mouse testis showed a strong mRNA signal at 3.5 kb and a weaker signal at 5.5 kb.  There are two possible explanations for this  discrepancy. In mouse sperm, testis, and epididymis, the STX-10 antigen may either (a)  64  be expressed but not detected immunologically or (b) not be expressed due to posttranslational modifications or regulatory elements.  STX-10 purified from HSA-10 immunoaffinity columns were analyzed by 10% SDS PAGE. In some of the polyacrylamide gels, several bands were observed at 75±5 kD. This may have been an indication of an impure antigen. If this same antigen were used to immunize mice or rabbits for antibody serum, this could explain the non-specificity of the clones sequenced at University of North Carolina, Chapel Hill, NC. These clones may also have been detected as positives due to technical error during initial immunoscreening experiments.  The phage containing the 1 kb fragment was used to transfect bacteria cells.  These  bacteria produced a fusion protein which was isolated, and then transferred onto nitrocellulose membranes. In these western blots, a protein of approximately 140 kD was observed. With the j3-galactosidase gene accounting for 114 kD of the fusion protein, the remaining 26 kD can be accounted for by the open reading frame of 168 amino acids (approximately 21 kD based on the average mass of an amino acid at 120 Daltons).  Foreign eDNA fragments are inserted in the unique EcoRI site of 2gt11 bacteriophage. However, this site may have been lost or modified in our clones during library cloning, since the positive controls (chicken ovalbumin cDNA and MSA-63 cDNA) were readily  65  cut by EcoRI. Instead, a double digestion with restriction enzymes KpnI and SstI was carried out.  Rabbit polyclonal anti-STX- 10 sera identified one positive clone. Generally, eDNA inserts isolated from immuno-screened clones do not contain the full length of the open reading frame (Snyder et al., 1987). In fact, the mRNA signal from BeWo cells was 5.5 kb in size. Therefore, the remaining 4.5 kb remains to be elucidated.  The 1 kb DNA fragment  contained only a short portion of the open reading frame. The majority of the fragment was composed of the untranslated 3’-end. In this region, homology was found between the 1 kb cDNA clone and a 449 bp clone isolated from the human male liver carcinoma Hep G2 cell line (Okubo et al., 1992). However, this cDNA fragment was found to be unique and its function still remains unknown. It is possible for two unrelated proteins to have similar or identical non-coding regions.  Once the gene for STX- 10 has been fully cloned and sequenced, analysis of its function, structure and control of gene expression can be studied in order to fully understand the role(s) of the STX- 10 antigen in the process of reproduction.  66  CONCLUSIONS  1  The STX- 10 antigen was confirmed to be a 75±5 kD protein present in human placenta and on the inner acrosome of human sperm.  2.  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Purification of an acrosomal antigen recognized by a monoclonal antibody and antifertility effects of isoimmune serum. International Journal of Andrology, 1989, 12: 451-463.  Liu MS, Chan K, Lau YF, and Lee CYG.  Molecular cloning of an acrosomal sperm  antigen gene and the production of its recombinant protein for immunocontraceptive vaccine. Molecular Reproduction & Development, 1990, 25: 302-308.  Liu MS, Aebersold R, Fann CH, and Lee CYG. Molecular and developmental studies of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.  Biology of  Reproduction, 1992, 46: 937-948.  Mathur S, Williamson HO, Landgrebe SC, Smith CL, and Fudenberg HI-I. Application of passive hemagglutination for evaluation of antisperm antibodies and a modified Coombs test for detecting male autoimmunity to sperm antigens.  Journal of Immunological  Methods, 1979, 30: 38 1-393.  Menge AC, Shoultz GK, Kelsey DE, Rutherford P, and Lee CYG. Characterization of monoclonal antibodies against human sperm antigens by immunoassays including sperm function assays and epitope evaluation. American Journal of Reproductive Immunology & Microbiology, 13: 108-114.  Metchnikoff E. Etudes sur la resorption de cellule. (Studies on the resorption of the cell) Annales de l’Institut Pasteur, 1899, 13: 73 7-739.  75  Moore 11DM, Smith CA, Hartman TD, and Bye AP. Visualization and characterization of the acrosome reaction of human spermatazoa by immunolocalization with monoclonal antibody. Gamete Research, 1987, 17:245-259.  Morton DB and McAnulty PA. The effect on fertility of immunizing female sheep with ram sperm acrosin and hyaluronidase. Journal of Reproductive Immunology, 1979, 1: 6172.  Myles DG and Primakoff P. Sperm proteins that serve as receptors for the zona pellucida and their post-testicular modification.  Annals New York Academy of Sciences, 1991,  486-493.  Myles DG, Hyatt H, and Primakoff P. Binding of both acrosome-intact and acrosome reacted guinea pig sperm to the zona pellucida during in vitro fertilization. Developmental Biology, 1987, 121: 559-567.  Naz RK.  The fertilization antigen (FA-1): applications in immunocontraception and  infertility in humans. American Journal of Reproductive Immunology & Microbiology, 1988, 16: 21-27.  Naz RK and Menge A. Development of antisperm contraceptive vaccine for humans: why and how? Human Reproduction, 1990a, 5: 511-518.  Naz RK.  Sperm surface antigens involved in mammalian feritlization: their role in  contraceptive vaccine development for humans. Current Opinion in Immunology, 1990b,  76  2: 748-751.  Okubo K, Hon N, Matoba R, Niiyama T, Fukushima A, Kojima Y, and Matsubara K. Large scale cDNA sequencing for analysis of quantitative aspects of gene expression. Nature Genetics, 1992, 2: 173-179.  O’Rand MG.  Inhibition of fertility and sperm-zona binding by antiserum to the rabbit  sperm membrane autoantigen RSA- 1. Biology of Reproduction, 1981, 25: 621-628.  O’Rand MG, and Irons GP.  Monoclonal antibodies to rabbit sperm autoantigens. II.  Inhibition of human sperm penetration of zona-free hamster eggs.  Biology of  Reproduction, 1984, 30: 73 1-736.  O’Rand M, Widgren EE, and Fisher SJ. Characterization of the rabbit spem membrane autoantigen RSA, as a lectin-like zona binding protein. Developmental Biology, 1988, 129: 23 1-240.  O’Rand MG, and Widgren EE. Molecular biology of a sperm antigen: Identification of the sequence of an autoantigenic epitope. Journal of Reproductive Immunology, 1989, supplement 6.  Polgar K, Yacono PW, Hill JA, Anderson DJ, Lee CYG, and Golan DE. Use of the translational mobility of a plasma membrane protein to assess fertilization of mouse oocytes and viability of mouse zygotes and two-cell embryos. Biology of Reproduction, 1994, 50: 474-480.  77  Primakoff P, Heaton MR, and Myles DG. Species specificity of hybridoma antibodies to surface antigens of guinea pig sperm. Gamete Research, 1983, 8: 385-394.  PrimakoffP, Hyatt H, and Myles DG. A role for the migrating sperm surface antigen PH20 in guinea pig sperm binding to the egg zona pellucida. Journal of Cell Biology, 1985, 101: 2239-2244.  Primakoff P, Cowan A, Hyatt H, Tredick-Kline J, and Myles DG.  Purification of the  guinea pig sperm PH-20 antigen and detection of a site-specific endoproteolytic activity in sperm preparations that cleaves PH-20 into two disulfide-linked fragments. Biology of Reproduction, 1988, 38: 921-934.  Primakoff P, Lathrop W, Woolman L, Cowan A, and Myles DG.  Fully effective  contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature, 1988, 335: 543-546.  Primakoff P, and Myles DG. Progress towards a birth control vaccine that blocks sperm function.  In: Gamete Interaction: Prospects for Immunocontraception.  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Anti-trophoblast monoclonal antibodies for  80  prenatal genetic diagnosis. In press.  Zaneveld LJD, De Jonge CJ, Amerson RA, and Mack SR. Human sperm capacitation and the acrosome reaction. Human Reproduction, 1991, 6: 1265-1274.  81  APPENDIX A  The sequences of the four newly synthesized oligonucleotide primers are as follows:  GL#5:  5 ‘-AGATCGTCTCCTTGCTGG-3’  from 289 bp to 306 bp  GL#6:  3’-CGAGGAGTTTTGATTGTCC-5’  from 826 bp to 808 bp  GL#7:  3 ‘-AACTTCGTCACCCAGGCC-5’  from 412 bp to 395 bp  GL#8:  3’-CGCTCGTTCAGTAGAACA-5’  from 703 bp to 686 bp  APPENDIX B  The following pages are the results of homology searches through GEN(]NFO) Blast:  82  I  NCBI BLAST E-Mail  ...,  7:03 PM 3/30/9...,Results-BLAST Server  Date: Wed, 30 Mar 1994 19:03:07 +0500 Subject: Results—BLAST Server Reply—To: “NCBI BLAST E-Mail Server” <blastencbi.nlm.nih. gov> From: NCBI BLAST E—Mail Server <blast@ncbi.nlm.nih.gov> Apparently—To: byl@ulam.generes.ca  To Obtain Documentation: send an e—mail message to ‘blast@ncbi.nlm.nih.gov’ with the word HELP in the body of the message. The documentation was last modified February 24th.  Trying muncher.., connected National Center for Biotechnology Information  (NCBI)  Experimental GENINFO(R)  (Muncher)  BLAST Network Service  Wed Mar 30 19:03:02 EST 1994,  Up 10:34,  load: 4.26,  5.38,  5.51  PEPTIDE SEQUENCE DATABASES nr Non—redundant PDB+SwissProt+PIR+SPupdate+GenPept+GPUpdate, updated daily for efficient, complete searches of the five component databases: pdb Brookhaven Protein Data Bank, October 1993 Release swissprot SWISS—PROT Release 28.0, March 1994 pir PIR Release 39.0 (complete), December 31, 1993 spupdate SWISS—PROT cumulative weekly update to the major release genpept CDS translations from GenBank(R) Release 81.0, Februa ry 15, 1994 gpupdate cumulative daily updates to the major release of genpept kabatpro Kabat Sequences of Proteins of Immunological Interest Releas e 5.0, August 1992 tfd TFD transcription factor (protein) database Release 7.0, Jun. 1993 acr * Ancient Conserved Region subset of SWISS—PROT, Dec. 3, 1993 palu * six—frame translations of representative human Alu repeats NUCLEOTIDE SEQUENCE DATABASES nr Non—redundant PDB+GBUpdate+GenBank+EmblUpdate+EMBL, updated daily for efficient, complete searches of the four component databases: pdb Brookhaven Protein Data Bank, October 1993 Releas e genbank GenBank(R) Release 81.0 (no daily updates), Febuary 15, 1994 gbupdate GenBank(R) cumulative daily updates to the major release embl EMBL Data Library, Release 37.0, December 1993 emblu EMBL Data Library cumulative daily updates to the major release vector Vector subset of GenBank(R), LANL, April 23, 1992 * repbase Human and other primate Alu repeats, Dr. Jerzy Jurka, Sept. 1993 kabatnuc Kabat Sequences of Nucleic Acid of Immunological Interes t Release 5.0, August 1992 epd Eukaryotic Promoter Database Release 35, June 1993 dbest ** Database of Expressed Sequence Tags Release 2.4, March 29, 1994 * **  Databases that are not accessible through the NCBI Retrieve E—mail server. dbEST data are available from est_report0ncbi.nlm .nih.gov. Send a HELP message to obtain instructions.  For a free subscription to “NCBI News”, the NCBI newsletter, send a request along with your name and postal mailing addres s to: infoncbi.nlm.nih.qov GenBank(R)  Release 81.0 is available via anonymous ftp on ncbi.nl m.nih.gov  All direct submissions of sequences to the GenBank(R) database, including those composed with Authorln, should be sent to the NCBI at any of the following addresses. If data is submitted on diskette, please indicat e whether Mac or PC.  83  1  [  NCBI BLAST E-Mail  Postal mail:  ...,  7:03 PM 3/3019...,Results-BLAST Server  GenBank Submissions National Center for Biotechnolog y In formation Building 38A, Room 8N—803 8600 Rockville Pike Bethesda, MD 20894—0001 Voice: 301—496—2475  E—mail submissions of new sequ ences: gbsub0ncbi.nlm.nih.qov E—mail submissions of updates: update@ncbi.nlm.nih.gov GenBank is a registered trademark of the National Institutes of Heal th. The help document for the BLAS T E—mail server was last modified Feb. 24th. BLASTN 1.3.13MP  (3—Mar—93)  (Build 14:38:36 Mar  3 1994)  Reference: Altschul, Stephen F., Warren Gish , Webb Miller, Eugene W. Myers, and David J. Lipman (1990) . Basic local alignment search tool. J. Mol. Biol. 215:403—410. Notice: this program is optimized to find nearly identical sequences rapid ly. To identify weak similarities encoded in nucleic acid, use BLAS TX or TBLASTN. Query  b2 (979 letters, both strands)  Database:  Searching  Non—redundant PDB+GBupdate+GenBank +EMBLupdate+EMBL, 5:09 AM EST Mar 30, 1994 167,244 sequences; 179,543,498 total letters. done  Highest—scoring Hit Extension vs. Count of Database Sequences Histogram units:  2190 Sequences  Highest Score Count——> I V 0 144480 Neighborhood word score thres hold, T 60 5349 61 2101 I: 62 1179 I: 63 723 I: 64 392 I: 65 2122 I: 66 1467 I: 67 1610 I: 68 695 I: 69 590 70 799 I: 71 755 I: 72 671 I: 73 445 I: 74 376 I: 75 515 I: 76 369 I: 77 306  : less than 2190 sequences  60  V  84  2  r  NCBI BLAST E-Mail  78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93  ...,  7:03 PM 3/30/9...,Results-BLAST Server  319 147 I: 231 I: 213 I: 188 I: 153 I: 250 I: 102 I: 71 I: 85 I: 75 1: 54 I: 55 I: 34 I: 42 I: 22 I:  94 95 96 97 98 99 100 101 102 103 104 105  32 23 23 20 10 10 10 7 6 5 3 2  I: 1: I: I: I: I: I: I: I: I: I: I:  106 107 108 109 110 111 112 113 114 115 116 117  1 72 2 3 5 1 0 3 0 2 1 9  I: I: I: I: I: I:  118 119 120  0  Expect  =  8.8e+02,  =  87., Observed  108  8.7,  9  Observed  =  259  I: I I: I: I: Expect  Observed  I  0 0 I  >>>>>>>>>>>>  121 122 123 124 125 126 127 128 129  0 0 0 0 0 0 2 0 0  130 131 132 133 134  0 I 1 I: 01 0 I Ol  Expect  =  4.9, Observed  <<<<<<<<<  9  =  I I I I I I: I 0.87, Observed  =  7  85  Cutoff Score 121  3  1  —  135 136 137 138 139 >139  0 1 0 0 4 1  A  VJ  I I: I I I: I:  High Score  Sequences producing High—scoring Segment Pairs: gb1D12242(HUM000S394 Human UepG2 3’—directed MboI cDN, c... Sequence 3 from patent US 4912038 Dog pulmonary surfactant protein 18  gbi1018631101863 gbIM1517OIDOGSP18A gb11003181100318 embl100318l100318  Smallest Poisson Probability  139 139  ...  (R06541A)  >gb1D122421HUM000S394 Human HepG2 3—directed MboI cDNA, Length 449  0.25  0.25  139  0.25  139  0.25  136 131 127  0.42 0.75 0.92  127  0.92  .  (C13O1A).  N  3.2e—166  2055  Sequence 3 from patent US 4933280 Nucleotide sequence 3 from patent nu... Caenorhabditis elegans cosmid C15H7 D.tigrina Dth—1 gene, exon 3  gbIZ22173ICEC15H7 gbIX692O1IDTDTH1E3 9b1D239561RICR06541A Rice cDNA, partial sequence gbID158O5IRIC13O1A Rice cDNA, partial sequence  P(N)  clone s394  Plus Strand HSPs: Score  2055  Identities Query:  (567.8 bits),  =  Query:  =  P  =  3.2e—166  411/411 (100%), Strand  =  Plus AGCC 622  11111)  I  Till  I  I  II  II  II  I  11111  11111  III  1 GATCAGAGACTGGAGAGGTGGAGTGAGAAGTCTCCGCTGCTCGGGCCCTCCTGGGG  I  AGCC 60  623 CCCGCTCCAGGGCTCGCTCCAGGACCTTCTTCACAACATGACTTGCTC GCTGTTACCTGC 682  11111111111111111 Sbjct:  3.2e—166,  563 GATCAGAGACTGGAGAGGTGGAGTGAGAAGTCTCCGCTGCTCGGGCCC TCCTGGGG  liii Sbjct:  Expect  411/411 (100%), Positives  liii) 11111111  II  111111111111111111111111  61 CCCGCTCCAGGGCTCGCTCCAGGACCTTCTTCACAAGATGACTTGCTCGCTGTTAC  CTGC 120  Query:  683 TTCCCCAGTCTTTTCTGAAAACTACAATTAGGGTGGGALPCCTCTGTA TTCCAAGG 742  Sbct:  121 TTCCCCAGTCTTTTCTGAAAAAtTACAAATTAGGGTGGGMAAGCTCTG TATTGAGAAGG 180  Query:  743 GTCATATTTGCTTTCTAGCAGGTTTGTTGTTTTGCCTGTTAGTTTTGA GGACCACGAAGC  111111111  liii  I  III  Ii  I  IllIllIll  III  I  1111111  1111111  802  111111111111111111111 liii 11111111111111111111111111111111111  Sbjct:  181 GTCATATTTGCTTTCTAGGAGGTTTGTTGTTTTGCCTGTTAGTTTTGAGGAGCAGC  Query:  803 TCATCGGGGCTTCTGTAGCCCCTCTCAAAAGGAGTCTTTATTCTGAGAATTTGCCTGA 862 111111111111111111111111111111111111111111111111111111111111  —  AAGC  240  Sbjct:  241 TCATGGGGGCTTCTGTACCCCCTCTCAAAACGAGTCTTTATTCTGAGA ATTTGAAGCTGA  Query:  863 AACCTCTTTAAATCTTCAGAATGATTTTATTGAAGAGGGCCGCAAGC CCCAAATGGAAAA 922  Sbct:  301 AACCTCTTTAAATCTTCAGAATGATTTTATTGAAGAGGGCCGCAAGCCCCAAATG GAAAA 360  Query:  923 CTCTTTTTAGAAMTATGMGTTTTTGATTGCTTTTGTATTTAATTCTGC  Sbjct:  111111111111111111111111111111111 I 11111111111111111111111111  11111111111 liii 111111111111111111111111111111 I 11111  973  361 CTGTTTTThGAAAATATGATGATTTTTGATTGCTTTTCTATTTAATTCTGC 411  86  300  From blast@ncbi.nlm.nih.gov Thu Mar 31 16:49:13 1994 Date: Thu, 31 Mar 1994 19:43:28 +0500 Sub:)ect: Results—BLAST Server Reply—To: “NCBI BLAST E—Mail Server” <blast@ncbi.nlm.nih.gov> From: NCBI BLAST E-Mail Server <blastncbi.nlm.nih.gov> Content—Length: 42321 To Obtain Documentation: send an e—mail message to ‘blast@ncbi.nlm.nih.gov’ with the word HELP in the body of the message. The documentation was last modified February 24th. Trying muncher... connected National Center for Biotechnology Information  (NCBI)  Experimental GENINFO(R) BLAST Network Service (Muncher) Thu Mar 31 19:43:03 EST 1994, Up 1 day,  11:14,  load: 50.83,  49.09,  48.92  PEPTIDE SEQUENCE DATABASES nr Non—redundant PDB+Swi s sProt+PIR+SPUpdate+GenPept+GPT.Jpdate, updated daily for efficient, complete searches of the five component databases: pdb Brookhaven Protein Data Bank, October 1993 Release swissprot SWISS—PROT Release 28.0, March 1994 pir PIR Release 39.0 (complete), December 31, 1993 spupdate SWISS—PROT cumulative weekly update to the major release genpept CDS translations from GenBank(R) Release 81.0, February 15, 1994 gpupdate cumulative daily updates to the major release of genpept kabatpro Kabat Sequences of Proteins of Immunological Interest Release 5.0, August 1992 tfd TFD transcription factor (protein) database Release 7.0, June 1993 acr * Ancient Conserved Region subset of SWISS—PROT, Dec. 3, 1993 palu * six—frame translations of representative human Alu repeats NUCLEOT IDE SEQUENCE DATABASES nr Non-redundant PDB+GBUpdate+GenBank+EmblUpdate+EMBL, updated daily for efficient, complete searches of the four component databases: pdb Brookhaven Protein Data Bank, October 1993 Release genbank GenBank(R) Release 81.0 (no daily updates), Febuary 15, 1994 gbupdate GenBank(R) cumulative daily updates to the major release embl EMBL Data Library, Release 37.0, December 1993 emblu EMBI, Data Library cumulative daily updates to the major release vector Vector subset of GenBank(R), LANL, April 23, 1992 repbase * Human and other primate Alu repeats, Dr. Jerzy Jurka, Sept. 1993 kabatnuc Kabat Sequences of Nucleic Acid of Immunological Interest Release 5.0, August 1992 epd Eukaryotic Promoter Database Release 35, June 1993 dbest ** Database of Expressed Sequence Tags Release 2.4, March 29, 1994 * **  Databases that are not accessible through the NCBI Retrieve E—mail server. dbEST data are available from est_report@ncbi.nlm.nih.gov. Send a HELP message to obtain instructions.  For a free subscription to “NCBI News”, the NCBI newsletter, send a request along with your name and postal mailing address to: info@ncbi.nlm.nih.gov GenBank(R) Release 81.0 is available via anonymous ftp on ncbi.nlm.nih.gov All direct submissions of sequences to the GenBank(R) database,  including  those composed with Authorln, should be sent to the NCBI at any of the following addresses. If data is submitted on diskette, please indicate whether Mac or PC. Postal mail:  GenBank Submissions National Center for Biotechnology Information Building 38A, Room 8N—803 8600 Rockville Pike Bethesda, MD 20894—0001 Voice: 301—496—2475  E—mail submissions of new sequences: gbsub@ncbi.nlm.nih.gov E—mail submissions of updates: update@ncbi.nlm.nih.gov GenBank is a registered trademark of the National Institutes of Health. The help document for the BLAST E—mail server was last modified Feb. 24th. BLASTP 1.3.11MP [29—Oct—93]  [Build 14:35:03 Mar  3 1994]  Reference: Altschul, Stephen F., Warren Gish, Webb Miller, Eugen e W. Myers, and David J. Lipman (1990). Basic local alignment search tool. J. Mol. Bid. 215:403—410. Query—  b2orf (168 letters)  Database:  Searching  Non—redundant PDB+SwissProt+SPupdate+PIR+GenPe pt+GPupdate, 5:02 AM EST Mar 31, 1994 113,405 sequences; 32,098,275 total letters. done  Highest—scoring Hit Extension vs. Count of Database Sequen ces Histogram units:  —  113 Sequences  Highest Score Count—-> V 0 1072 I———— Neighborhood word score threshold, T 11 506 12 907 13 986 14 1253 15 1419 16 1722 17 2311 18 3357 1 9 37 47 20 4325 21 5273 22  55 63  23 24 25 26 27  6340 7326 7359 77 62 7266  —  : less than 113 sequences  11  sas____a_______a  _a____fln_____  annnaana=nsnsnnnnnnn  nnnaas  1 flaaasnanflaflflaaaasaaaaaanflnflflflflaflfl 1 1 Sflstflflflflflflflflflflflflflflfl aaflflflflflflflflflflf  lfl  ci  28 29 30 31 32 33 34 35 36 37 38 39 40 41  6842 6290 6262 4692 4458 3678 2951 2195 1538 1368 1050 837 615 476  42 43 44 45 46 47 48  351 285 297 209 133 98 55  I: I:  49 50 51 52 53 54 55  40 24 30 50 30 9 16  I: I: I: I: I: I: I:  56 57  13 I: 0 I  I I I I————=————————————————— I I  I I I———— Expect  —  8.4e+02, Observed  Expect  —  85., Observed  Expect  —  8.7, Observed  Expect  —  4.5,  1659  I— I—  I  58 59 60 61 62  2 3 0 2 0  I: I  63 64 65 66 67 68 69  0 2 2 0 0 0 0  I I: I: I I I I  —  231  —  >>>>>>>>>>>>>  Observed  32 19  —  <<<<<<<<<  I:  Cutoff Score 58  I Expect  Expect  70 71 >71  —  0.88,  —  Observed  0.089, tbserved  12  =  —  8  0 I 4 I: 4 I:  Sequences producing High—scoring Segment Pairs: pirIS31908IS31908 gpIM17423ISCMPRSM_1 pir1A601451A60145  tiemoglobinase fluke (Schistosoma... S.niansoni protease gene, complete hemoglobinase (EC 3.4.—.—) precurs... —  89  High Score 123 122 121  Smallest Poisson Probability P(N) N 3.6e—09 4.7e—09 7.le—09  1 1 1  5pIPO9841IJIGLBSCHMA pirB40171B40171 5pIP19492IGLRC RAT pirIJHO3li.IJHO311 gpM85036IRATGLUR3A_1 gpIM848O3IHPENBP1_1 spIP31853ATPX SPIOL SpIP3253IRRPP:PIHD gpIX14459IDMBCD26B_1 5pIPO9O81IHMBC_DROME pirIC35815C35815 pir1D358151D35815 pir1A358151A35815 pir1B358151B35815 5pIQO1134IKICH_RAT pir1S354011S3540i pirIA43336A43336 sp{P3O622IREST HUMAN gpIX53155IDMMHCHIN_7 gpIM61229IDROMHC_1 gpIM61229IDROMHC_2 gpU05823IMMU058231 pir1A324911A32491 pir1B324911B32491 pir1S357601535760 gpIXO6826IATVIRB_3 spIPO535OIVIB1AGRT9 5pIP32532IRRPP_PI1HE gpIM73O45IDOGSNVD17A1 gpID25278IHUMORFO_l gpIS66910IS66910_l pir1S289l61528916 gpIM82829IHUNORFD_l spIPO8799IMYS2DICDI gpIZ21513IRNINNEGLA_1 gpIXO7278IDMLANDMO1 spIQO592OIPYC MOUSE sp1P29415)CX56 CHICK pir1A453381A45338 pirID60110D60li0  HEMOGLOBINASE PRECURSOR (EC 3.4.21... glutamate receptor 3 precursor rat GLUTAMATE RECEPTOR C PRECURSOR (GL... glutamate receptor K3 chain precur... glutamate receptor subunit 3 [Ratt... nucleotide binding protein [Hepati... ATP SYNTHASE B’ CHAIN PRECURSOR (E... RNA POLYMERASE ALPHA SUBUNIT (EC 2... Drosophila c53.46.9 bcd (bicoid) in... HOMEOTIC BICOID PROTEIN (PRD-4). >... myosin heavy chain 3, muscle fru... myosin heavy chain 4, muscle fru... myosin heavy chain 1, muscle fru... myosin heavy chain 2, muscle fru... CHOLINE KINASE Ri (EC 2.7.1.32). >... N protein type 1 Streptococcus p... microtubule—vesicle linker CLIP—17... RESTIN (CYTOPLASMIC LINKER PROTEIN... D.melanogaster muscle myosin heavy... myosin heavy chain [Drosophila mel... myosin heavy chain [Drosophila mel... pericentrin [Mus musculus] myosin heavy chain 1, muscle fru... myosin heavy chain 2, muscle fru... fcrA protein precursor Streptoco... Agrobacterium tumefaciens virB ope... VIRB1 PROTEIN PRECURSOR. >pirISOO7... R1A POLYMERASE ALPHA SUBUNIT (EC 2... Dog inserted sequence in spleen ne... ORF [Homo sapiens] BRG1 [Homo sapiens] dystrophin mouse >gpIM68859IMUSD... fusion protein [Homo sapiens] MYOSIN II HEAVY CHAIN. >pir1A26655... integral membrane glycoprotein [Ra... Drosophila rnRNA for nuclear lamin ... PYRUVATE CARBOXYLASE (EC 6.4.1.1) ... GAP JUNCTION CX56 PROTEIN (CONNEXI... connexin—56 chicken >gpIL02838IC... repetitive protein antigen 3 Try... —  —  —  —  —  —  —  —  —  —  —  —  110 71 7]. 71 71 44 49 47 65 65 53 53 53 53 42 50 64 64 53 53 53 41 53 53 38 61 61 47 59 49 43 47 59 51 59 44 48 58 58 47  2.8e—07 0.11 0.11 0.11 0.11 0.37 0.38 0.55 0.56 0.56 0.60 0.60 0.61 0.61 0.61 0.70 0.71 0.71 0.75 0.79 0.79 0.82 0.86 0.86 0.90 0.93 0.93 0.95 0.990 0.993 0.996 0.997 0.998 0.998 0.998 0.98 0.999 0.9997 0.9997 0.9997  >pirjS31908S319O8 hemoglobinase fluke (Schistosoma japonicum) >gpIX7O967ISJRNAHEMO_l hemoglobinase [Schistosoma japonicum) Length — 423 —  Score 123 (56.8 bits), Expect = 3.6e—09, P 3.6e—09 Identities — 26/84 (30%), Positives — 48/84 (57%)  Query: Sbj Ct:  Query: Sbjct:  4 QYGNKTISTMKVMQFQGMKRKASSPVPLPPVTHLDLTP SPDVPLTIMKPKLMNTNDLEES 63 +YG+K + + + +FQG ++KAS+ D PS D+PL + R++M N++ + PP+ 268 RYGDKKMGKLYLSEFQGSRKKASTEHDEPPMKPKDS IP SRDIPLHTLHRRIMMANNMNDK 327 64 RQLTEEIQRHLDARHLIEKSVRKI 87 L++ L RLI+++ I 328 TLLMKILGLKLKRRDLIKDTMEVI 351  >gpIM17423ISCMPRSM_1 S.mansoni protease gene, complete cds. [Schistosoma mansoni] >gpIM17423ISCMPRSM_1 protease [Schistosoma rnansoni]  90  1 1 1 1 1 2 2 2 1 1 2 2 2 2 3 3 1 1 2 2 2 4 2 2 3 1 1 2 1 3 3 2 1 2 1 2 2 1 1 2  Length  —  353  Score — 122 (56.4 bits), Expect — 4.7e—09, P 4.7e—09 Identities = 29/92 (31%), Positives 50/92 (54%)  Query: Sbj ct:  Query: Sbjct:  4 QYGNKTISTMKVMQFQGMKRKASSPVPLPPVTHLDLTPSPDVPLTflil(RKLMNTNDLEES 63 +YG+ + + V +FQG + K+SS PP+ S D+PL + R++M TN+ E+ 198 RYGDTRNGKLYVSEFQGSRDKSSSENDEPPMI(PRISVASRDIPLHTLHRQIMNTNNAEDK 257 64 RQLTEEIQRHLDARHLIEKSVRKIVSLLAASE 95 L++ L RLIE+++IV++ E 258 SFLMQIIGLKLKPRDLIEDT(LIVKVMNNEE 289  >pir1A601451A60145 hemoglobinase man soni) Length — 429  (EC 3.4.—.—) precursor  —  fluke  (Schistosoma  Score = 121 (55.9 bits), Expect — 7.le—09, P 7.le—09 Identities — 29/92 (31%), Positives 50/92 (54%) Query: Sbj ct: Query: Sbj ct:  4 QYGNKT ISTMKVMQFQGMKRKASSPVPLPPVTHLDLTPSPDVPLTIMKRKLMNTNDLEES 63 +YG+ + + V +FQG + K+SS S D+PL + R++M TN+ E+ PP+ 274 RYGDTRNGKLYVSEFQGSRDKSSSENDEPPMKPRHS IASRDIPLHTLHRQIMMTNNAEDK 333 64 RQLTEEIQRHLDARHLIEKSVRKIVSLLAASE 95 L + + L R LIE +++ IV ++ E 334 SFLMQILGLKLKRRDLIEDTMKLIVKVMNNEE 365  >spIPO9841IHGLBSCHMA HEMOGLOBINASE PRECURSOR (EC 3.4.21.—) (ANTIGEN SM32). >gpIM213O8ISCMHGBA1 S.mansoni hemoglobinase (Sm32) mRNA, complete cds. [Schistosoma rnansoni3 >gpM21308 ISCMHGBA_1 hemoglobinase [Schistosoma mansoni] Length — 429 Score — 110 Identities Query: Sbj ct: Query: Sbjct:  (50.8 bits), Expect 2.8e—07, P — 2.8e—07 27/92 (29%), Positives 49/92 (53%)  4 QYGNKTISTMKVMQFQGMKRKASSPVPLPPVTHLDLTP SPDVPLTIMKRKLMNTNDLEES 63 +YG+ + V +FQG + K+S+ + P+ S D+PL ÷ R++M TN+ E+ 274 RYGDTRNGKLYVSEFQGSRDKSSTENDESPMKPRHS IASRDIPLHTLHRQIMMTNNAEDK 333 64 RQLTEEIQRHLDARHLIEKSVF<XIVSLLAASE 95 L + + L R LIE +++ IV ++ E 334 SFLMQILGLKLKRRDLIEDTMKLIVKVMNNEE 365  >pirIB4Ol7lIB4Ol7l glutamate receptor 3 precursor Length 886 Score = 71 Identities Query: Sbjct:  —  rat  (32.8 bits), Expect 0.12, P — 0.11 14/43 (32%), Positives 27/43 (62%)  49 IMKRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLL 91 + R + N D++E R++ EE+ R + R+LI+ V +1 ++L 182 VTARSVGNIKDVQEFRRIIEEMDRRQEKRYLIDCEVERINTIL 224  >sp1Pl9492 GLRC RAT GLUTAMATE RECEPTOR C PRECURSOR (GLUR-C) (GLUR-3) >pir)C401701C40170 glutamate receptor C precursor rat —  9’  (GLUR-K3).  ci >gpIM3642OIRATGLURC1 Rat glutamate receptor (G1uR-C) mRNA, complete cds. [Rattus norvegicus] Length = 888 Score — 71 Identities Query: Sbjct:  (32.8 bits), Expect — 0.12, P 0.11 14/43 (32%), Positives 27/43 (62%)  —  49 IMKP.KLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLL 91 + R + N D++E R++ EE+ R + R+LI+ V +1 ++L 182 VTARSVGNIKDVQEFRRIIEEMDRRQEKRYLIDCEVERINTIL 224  >pirIJHO311IJHO311 glutamate receptor K3 chain precursor rat >gpIM38062IRATAMPASGC1 ANPA selective glutamate receptor [Rattus norvegicus] >gpjX54656RNGLURK3l glutamate receptor [Rattus norvegicus] Length — 888 —  Score — 71 Identities Query: Sbjct:  (32.8 bits), Expect 0.12, P 0.11 14/43 (32%), Positives = 27/43 (62%)  49 IMKRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVKIVSLL 91 + R + N D++E R++ EE+ R + R+LI+ V +1 ++L 182 VTARSVGNIKDVQEFRRIIEEMDRRQEKRYLIDCEVERINTIL 224  >gpIM85O36IRATGLUR3A_l glutamate receptor subunit 3 Length 888 Score — 71 Identities  Query: Sbjct:  (32.8 bits), Expect = 0.12, P — 0.11 14/43 (32%), Positives 27/43 (62%)  —  49 IMKRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLL 91  + R + N D++E R++ EE+ R + R+LI+ V +1 ++L 182 VTARSVGNIKDVQEFRRIIEEMDRRQEKRYLIDCEVERINTIL 224  >gpM84803HPENBPl_1 nucleotide binding protein Length 141 Score 44 Identities Query:  Sbjct:  Sbjct:  [Hepatitis E virus]  (20.3 bits), Expect 0.47, Poisson P(2) 8/20 (40%), Positives 11/20 (55%)  0.37  27 SPVPLPPVTHLDLTPSPDVP 46 +P PP+ LD+PP P 77 TPAAPPPLLALDPSPPPSAP 96  Score — 44 Identities Query:  [Rattus norvegicus)  (20.3 bits), Expect 0.47, Poisson P(2) 9/20 (45%), Positives 12/20 (60%) —  —  0.37  —  24 KASSPVPLPPVTHLDLTPSP 43 +A++PP P LLPSP 72 RAATPTPAAPPPLLALDPSP 91  >spIP31853IATPX_SPIOL ATP SYNTHASE B’ CHAIN PRECURSOR (EC 3.6.1.34) (SUBUNIT II). >pir15344731S34473 H+-transporting ATP synthase (EC 3.6.1.34) chain 9 spinach >gpX7l397ISTATPGMRl CF(o)II ATP synthase subunit 9 [Spinacia oleracea] Length — 222 —  92  Score — 49 Identities Query: Sbjct:  Sbjct:  (22.6 bits), Expect 1.4e+02, P 11/23 (47%), Positives 12/23  1.0 (52%)  25 ASSPVPLPPVTHLDLTPSPDVPL 47 ASS LP T +TPP PL 7 ASSSKTLPTTTTTTITPKPKFPL 29  Score — 45 Identities Query:  ci (20.8 bits), Expect — 0.48, Poisson P(2) 9/22 (40%), Positives 13/22 (59%)  0.38  —  27 SPVPLPPVTHLDLTPSPDVPLT 48 SP LPP+ HL+L+ 38 SPPQLPPLKHLNLSVLKSAAIT 59  >spIP32531IRRPP_PI1HD RNA POLYMERASE ALPHA SUBUNIT (EC 2.7.7.48) (NUCLEOCAPSID PHOSPHOPROTEIN). >pirIC4O234IRRNZ73 polymerase—associated nucleocapsid phosphoprotein parainfluenza virus type 1 (strain CI—5/73) Length 568 —  Score — 47 Identities Query:  Sbjct:  58 NDLEESRQLTEEIQRHLDARHLIEK 82 N +EE+R L ++IQ +D± + K 384 NKVEENRTLLKQIQEEIDSSRDLHK 408  Score — 46 Identities  Query: Sbjct:  (21.7 bits), Expect — 3.le+02, P 1.0 9/25 (36%), Positives 16/25 (64%)  —  (21.2 bits), Expect 0.80, Poisson P(2) 9/38 (23%), Positives — 20/38 (52%)  —  0.55  —  34 VTHLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQ 71 +T + PSP++ +++RK ++ S+ +E Q 208 ITDVITNPSPELEDAVLQRKKRRPTTIKRSQTRSERTQ 245  >gpX14459IDMBCD26B1 Drosophila c53.46.9 bcd (bicoid) mRNA (major 2.6 kb transcript) [Drosophila melanogaster] Length — 477 Score = 65 Identities Query:  (30.0 bits), Expect 0.81, P 0.56 17/62 (27%), Positives 31/62 (50%)  —  Sbjct:  38 DLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRXIVSLLAASE.AE 97 D+ PS ++P +++ R+ S Q+ E Q L R+L T + + + LA A+ 81 DVFPSEELPDSLVMPRPRRTRTTFTSSQIAELEQHFLQGRYLTAPRLADLSAKLALGTAO 140  Query:  98 VE99  Sbjct:  141 VK 142  >5pIPO9O81IHMBCDROME HOMEOTIC BICOID PROTEIN (PRD—4). >pirISOO835IWJFFBC homeotic protein bicoid fruit fly (Drosophila melanogaster) >gpIXO787OIDMBCDG_l bcd gene product [Drosophila melanogaster] Length — 494 —  Score — 65 Identities Query:  (30.0 bits), Expect — 0.82, P — 0.56 17/62 (27%), Positives — 31/62 (50%)  —  38 DLTPSPDVPLTIMKR!CLMNTNDLEESRQLTEE IQRHLDARHLIEKSVRKIVSLLAASEAE 97  93  Sbj Ct:  D+ PS ++P +++ R+ T S Q+ E Q L R+L + + + LA A+ 81 DVFPSEELPDSLVMIP.PRRTRTTFTSSQIAELEQHFLQGRYLTA2RLADLSAKLALGTAQ 140  Query:  98 yE 99  Sbjct:  141 VK 142  >pirC35815IC35815 myosin heavy chain 3, muscle melanogaster) (fragment) Length — 1175 Score — 53 Identities Query: Sbjct:  Sbjct:  Sbjct:  (24.5 bits), Expect — 44., P — 1.0 13/37 (35%), Positives — 20/37 (54%)  —  (21.7 bits), Expect 0.92, Poisson P(2) 16/58 (27%), Positives 25/58 (43%)  (20.3 bits), Expect 6.5, Poisson P(2) 11/37 (29%), Positives 19/37 (51%)  65 QLTEEI QRHLDARHLIEKSVRKIVSLLAASEAEVEQL 101 +L + ++R R +EKS RK+ L ++ V L 271 ELEDSLEREKKVRGDVEKSKRKVEGDLKLTQEAVADL 307  Score 53 Identities  Sbjct:  Sbj ct:  Sbj ct:  fruit fly (Drosophila  (24.5 bits), Expect 44., P 1.0 13/37 (35%), Positives 20/37 (54%)  (21.7 bits), Expect 0.92, Poisson P(2) 16/58 (27%), Positives — 25/58 (43%)  0.60  —  36 HLDLTPSPDVPLTIMKRXLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAA 93 +LL P +L+N++E+ L EE + + H E VRK+ LA 59 YLQLRTWPWYKLWQKVKPLLNVSRIEDEIARLEEKAI(KAEELHAAEVKVRXELEALNA 116  Score — 44 Identities  Query:  —  61 EESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAE 97 +L E +RH DA+ + KS R++ L E SE + 1059 ELENELDGEQRRHADAQKNLRXSERRVKELSFQSEED 1095  Score — 47 Identities Query:  1.0  —  >pir1D358151D35815 myosin heavy chain 4, muscle melanogaster) (fragment) Length — 1175  Query:  0.60  36 HLDLTP SPDVPLTIMKRXLMNTNDLEESRQLTEE IQRHLDARHLIEKSVRKIVSLLAA 93 +LL P +L+N++E+ L + + H E VRX+ LA EE 59 YLQLRTWPWYKLWQKVKPLLNVSRIEDEIARLEEKAKKAEELHAAEVKVRKELEALNA 116  Score 44 Identities  Query:  fruit fly (Drosophila  61 EESRQLTEEIQRHLDARI-ILIEKSVRKIVSLLAASEAE 97 E +L E +RH DA+ + KS R++ L SE + 1059 ELENELDGEQRRHADAQKNLRKSERRVKELSFQSEED 1095  Score — 47 Identities Query:  —  (20.3 bits), Expect — 6.5, Poisson P(2) 11/37 (29%), Positives — 19/37 (51%)  —  1.0  —  65 QLTEEI QRHLDARHLIEKSVPKIVSLLAASEAEVEQL 101 +L + ++R R +EKS PK+ L ++ V L 271 ELEDSLEREKKVRGDVEKSKRKVEGDLKLTQEAVADL 307  >pir1A358151A35815 myosin heavy chain 1,  muscle  —‘.4-  —  fruit fly (Drosophila  ,  94  ci melanogaster) Length = 1201 Score — 53 Identities Query: Sbjct:  Sbj Ct:  Sbjct:  (21.7 bits), Expect — 0.94, Poisson P(2) 16/58 (27%>, Positives 25/58 (43%)  (20.3 bits), Expect 6.6, Poisson P(2) 11/37 (29%), Positives — 19/37 (51%)  melanogaster) Length = 1201 Score — 53 Identities  Sbjct:  Sbj ct:  Sbjct:  —  fruit fly (Drosophila  (fragment)  (24.5 bits), Expect 45., P — 1.0 13/37 (35%), Positives 20/37 (54%)  —  (21.7 bits), Expect — 0.94, Poisson P(2) 16/58 (27%), Positives 25/58 (43%)  0.61  —  36 HLDLTPSPDVPLTIMI(RKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAA 93 +LL P L +L+N++E+ EE + + H E VRK+ LA 59 YLQLRTWPWYKLWQKVKPLLNVSRIEDEIARLEEKAEKAEELHAAEVKVRXELEALNA 116  Score — 44 Identities Query:  1.0  61 EESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAE 97 +L E +RH DA+ + KS R++ L E SE + 1059 ELENELDGEQP.RHADAQKNLRKSERRVKELSFQSEED 1095  Score = 47 Identities Query:  —  65 QLTEEIQRHLDARHLIEKSVRKIVSLLAASEAEVEQL 101 +L + ++R R +EKS RK+ L ++ V L 271 ELEDSLEREKKVRGDVEKSKRI(VEGDLKLTQEAVADL 307  >pir1B358151B35815 myosin heavy chain 2, muscle  Query:  0.61  —  —  36 HLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQRRLDARHLIEKSVRKIVSLLAA 93 +LL + + H E VRK+ LA P L +L+N++E+ EE 59 YLQLRTWPWYKLWQKVKPLLNVSRIEDEIARLEEKAI(KAEELHAAEVKVRXELEALNA 116  Score — 44 Identities Query:  (24.5 bits), Expect 45., p — 1.0 13/37 (35%), Positives — 20/37 (54%)  —  61 EESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAE 97 +L E +RH DA+ + KS R++ L E SE + 1059 ELENELDGEQRRHADAQKNLRKSERRVKELSFQSEED 1095  Score — 47 Identities Query:  (fragment)  (20.3 bits), Expect 6.6, Poisson P(2) 11/37 (29%), Positives 19/37 (51%)  =  1.0  —  65 QLTEEI QRHLDARHLIEKSVRKIVSLLAASEAEVEQL 101 +L + ++R R +EKS RK+ L ++ V L 271 ELEDSLEREKKVRGDVEKSKRKVEGDLKLTQEAVADL 307  >SpIQO1134IKICH_RAT CHOLINE KINASE Ri (EC 2.7.1.32). >gpjD1O262IRATCKR_1 choline kinase Ri [Rattus norvegicus] Length — 435 Score — 42 Identities Query: Sbjct:  (19.4 bits), Expect 1.5e+03, P — 1.0 9/19 (47%), Positives — 10/19 (52%)  —  29 VPLPPVTHLDLTPSPDVPL 47 +PPP LLPP PL 56 LPPPPPPPLPLPPPPSPPL 74  95  Page  10 Score — 41 Identities  Query: Sbjct:  Sbjct:  1.0  17 QFQGMKRKASSPVPLPPVTHLDLTPSP 43 L PSP Q G + +PPPP 46 QLGGRSQPLALPPPPPPPLPLPPPPSP 72  Score — 38 Identities Query:  (18.9 bits), Expect — 16., Poisson P(2) 10/27 (37%), Positives — 12/27 (44%)  —  (17.6 bits), Expect 0.95, Poisson P(3) 8/22 (36%), Positives = 15/22 (68%)  0.61  —  80 IEKSVRKIVSLLAASEAEVEQL 101 +EK + +++ L + EA V+QL 231 MEKYLNQVLRLKFSREARVQQL 252  >pir1S354011S35401 M protein type 1 Streptococcus pyogenes >gpIX62131ISPEMM1M_1 M protein type 1 [Streptococcus pyogenes) Length = 484 —  Score 50 Identities Query: Sbjct:  Sbj ct:  Sbjct:  Sbj ct:  Sbj ct:  (18.0 bits), Expect 4.le+03, P — 1.0 9/21 (42%), Positives 14/21 (66%)  =  (18.0 bits), Expect 64., Poisson P(2) 11/35 (31%), Positives = 18/35 (51%)  1.0  70 IQRHLDARHLIEKSVRKIVSLLAASEPEVEQLLSE 104 ++R LDA + +K V K + A +E+L E 329 LRRDLDASREAKKQVEKALEEANSKLAALEKLNKE 363  Score 38 Identities Query:  1.0 (57%)  89 SLLAASEAEVEQLLSERAPLT 109 +LL + E++QL SE+ LT 205 NLLGNRKLELDQLSSEKEQLT 225  Score 39 Identities Query:  (18.5 bits), Expect 3.Oe+03, P 10/33 (30%), Positives — 19/33  =  68 EEIQRHLDARHLIEKSVRXIVSLLAASEAEVEQ 100 + ++R LDA +K V K ++ L A +V++ 243 QSLRRDLDASREAKKQVEKDLANLTAELDKVKE 275  Score = 39 Identities Query:  1.0 (67%)  —  59 DLEESRQLTEEIQRHLDARHLIEKSVRK 86 +LEES++LTE+ + L A+ E +V K 363 ELEESKKLTEKEKAELQAKLEAEANVLK 390  Score — 40 Identities  Query:  (23.1 bits), Expect = 1.le+02, P 12/28 (42%), Positives = 19/28  —  (17.6 bits), Expect 1.2, Poisson P(3) 8/40 (20%), Positives — 20/40 (50%)  0.70  =  38 DLTPSPDVPLTINKRKLMNTNDLEESRQLTEEIQRHLDAR 77 + + + DL +LE+++Q E+ ++ L+ + R 72 DLKARLENAMEVAGRDFKRAEELEKAKQALEDQRXDLETK 111  >pir)A433361A43336 microtubule—vesicle linker CLIP—170 human >gpIM975O1IHTJMCLIP_1 cytoplasmic linker protein—170 alpha—2 sapiens] Length — 1392 —  (: I  ,J -  96  [Homo  Score — 64 Identities Query: Sbjct:  Query: Sbjct:  (29.6 bits), Expect — 1.2, P — 0.71 17/68 (25%), Positives — 38/68 (55%)  —  51 KRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAPLTG 110 +R ++N LE ++ ++ 1+ + + ++KS+ +LL +AE+E+L +E L G 1156 ERSVLNNQLLEMKKRESKFIKDADEEKASLQKSISITSALLTEKDAELEKLRNEVTVLRG 1215  111 ESCYPEAL 118 + ++L 1216 ENASAKSL 1223  >spIP3O622IREST HUMAN RESTIN (CYTOPLASMIC LINKER PROTEIN—170 ALPHA-2) (CLIP—170). >pir1S226951S22695 restin human >gpIX64838IHSPESTIN_1 restin [Homo sapiens] Length — 1427 —  Score — 64 Identities Query: Sbct: Query: Sbjct:  (29.6 bits), Expect — 1.2, P — 0.71 17/68 (25%), Positives 38/68 (55%)  =  51 KRKLMNTNDLEESRQLTEEI QRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSEP.APLTG 110 +R ++N + + ++KS+ LE ++ ++ 1+ +LL +AE+E+L +E L G 1 191 ERSVLNNQLLEMKKRESKFIKDADEEKASLQKSI SITSALLTEKDAELEKLRNEVTVLRG 1250 111 HSCYPEAL 118 + ++L 1251 ENASAKSL 1258  >gpIX53155IDMMHCHIN_7 D.melanogaster muscle myosin heavy chain gene (exon 12 to 19) alternatively spliced products [Drosophila melanogaster] Length — 302 Score — 53 Identities Query: Sbj ct:  61 EESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASE.AE 97 E +L E +RH DA+ + KS R++ L SE + 187 ELENELDGEQRRHADAQKNLPKSEPRVKELSFQSEED 223  Score — 44 Identities Query: Sbjct:  (24.5 bits), Expect — 40., P — 1.0 13/37 (35%), Positives 20/37 (54%)  —  (20.3 bits), Expect — 1.4, Poisson P(2) 9/15 (60%), Positives = 11/15 (73%)  —  0.75  =  58 NDLEESRQLTEEIQR 72 N+LEESR L E+ R 49 NELEESRTLLEQADR 63  >gplM61229DROMHC_1 niyosin heavy chain [Drosophila melanogaster] Length — 1962 Score — 53 Identities Query: Sbj Ct:  (24.5 bits), Expect — 45., P — 1.0 13/37 (35%), Positives — 20/37 (54%)  —  61 EESRQLTEEI QRHLDARHLIEKSVRKIVSLLAASEAE 97 +L E +RH DA+ + KS R++ L E SE + 1820 ELENELDGEQRRHADAQKNLRKSERRVKELSFQSEED 1856  Score — 47 Identities  (21.7 bits), Expect = 1.6, Poisson P(2) 16/58 (27%), Positives — 25/58 (43%)  —  97  —  0.79  Page  12J Query:  Sbjct:  36 HLDLTPSPDVPLTIM1(RKLMNTNDLEESRQLTEEIQRRLDARHLIEKSVR(IVSLLAA 93 +LL P L +L+N++E+ EE + + H E VRK+ LA 820 YLQLRTWPWYKLWQK LLNVSRIEDEIARLEEKAKKAEELHAAEVKVRKELEALNA 877  >gpIM61229DROMHC_2 rnyosin heavy chain [Drosophila melanogaster] Length — 1962 Score 53 Identities Query: Sbjct:  61 EESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAE 97 +L E +RH DA+ + KS R++ L E SE + 1820 ELENELDGEQRRHADAQKNLRKSERRVKELSFQSEED 1856  Score 47 Identities Query:  Sbj ct:  (24.5 bits), Expect — 45., P — 1.0 13/37 (35%), Positives 20/37 (54%)  =  1.6, Poisson P(2) (21.7 bits), Expect 16/58 (27%), Positives — 25/58 (43%)  36 HLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRXIVSLLAA 93 +LL P L +L+N++E+ EE + + H E VRK+ LA 820 YLQLRTWPWYKLWQKVKpLLNVSRIEDEIARLEEKAK1(ELHAAEVKVR1CELEALNA 877  >gp1U05823(MMU058231 pericentrin Length 1920 Score — 41 Identities Query:  Sbjct:  Sbj ct:  Sbjct:  Sbjct:  =  1.0  (18.9 bits), Expect 75., Poisson P(2) 9/30 (30%), Positives — 18/30 (60%)  1.0  —  (18.0 bits), Expect — 8.0, Poisson P(3) 11/46 (23%), Positives 22/46 (47%)  1.0  —  53 KLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAEV 98 K + N E RQL ++ ++ H + + ++ +SL+ EV 1441 KSLIENLQENQRQLQKDKAEEIEQLHEVIEKLQSELSLMGPKVHEV 1486  Score — 37 Identities Query:  (18.9 bits), Expect = 75., Poisson P(2) 9/30 (30%), Positives — 17/30 (56%)  =  49 IMKRI(LMNTNDLEESRQLTEEIQRHLDARH 78 +++ L ++ LEE+RQL ++R R+ 1045 LLEMALDSSKQLEEARQLHRCVEREFRHPN 1074  Score — 39 Identities Query:  [Mus niusculus]  75 DARHLIEKSVRKIVSLLAASEAEVEQLLSE 104 +A EAEVE+S+ DA + ++++ 1549 DAEEVAARHLAELEHCVALREAEVEANASQ 1578  Score — 41 Identities Query:  0.79  —  (17.1 bits), Expect — 1.7, Poisson P(4) 9/35 (25%), Positives 17/35 (48%)  —  0.82  38 DLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQR 72 + + +L S D + +LEE R E++Q+ 866 ELQASQDQVAQVRDKVFLLNRELEECRADVEQLQQ 900  >pir1A324911A32491 myosin heavy chain 1, muscle melanogaster) Length — 2385 Score — 53 Identities  —  (24.5 bits), Expect — 45., P — 1.0 13/37 (35%), Positives = 20/37 (54%)  —  98  fruit fly  (Drosophila  Page  13 Query: Sbj ct:  61 EESRQLTEEIQRHLDARHLIEKSVPKIVSLLAASEAE 97 E +L E +RH DA+ + KS R++ L SE + 2269 ELENELDGEQRRHADAQKNLRKSERRVKELSFQSEED 2305  Score — 47 Identities Query: Sbj Ct:  (21.7 bits), Expect — 1.9, Poisson P(2) 16/58 (27%), Positives 25/58 (43%)  36 HLDLTPSPDVPLTIMKPKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAA 93 +LL P +L+N++E+ L EE + + H E VRK+ LA 1243 YLQLRTWPWYKLWQKVKPLLNVSRIEDEIARLEEKAKKPEELHAAEVKVRKELEALNA 1300  >pir1B324911B32491 myosin heavy chain 2, imscle melanogaster) Length = 2411 Score 53 Identities Query: Sbj ct:  Sbj Ct:  —  fruit fly (Drosophila  (24.5 bits), Expect 45., P — 1.0 13/37 (35%), Positives = 20/37 (54%)  —  61 EESRQLTEEI QRHLDARHLIEKSVRKIVSLLAASEAE 97 E +L E +RH DA+ + KS R++ L SE + 2269 ELENELDGEQPRHADAQKNLIKSERRVKELSFQSEED 2305  Score — 47 Identities  Query:  0.86  —  (21.7 bits), Expect — 2.0, Poisson P(2) 16/58 (27%), Positives 25/58 (43%)  0.86  —  36 HLDLTP SP DVPLTIMKPKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAA 93 +LL P +L+N++E+ L EE + + H E VRK+ LA 1243 YLQLRTWPWYKLWQKVKPLLNVSRIEDEIARLEEKAKKAEELHAAEVKVRKELEALNA 1300  >pirS35760IS35760 fcrA protein precursor Streptococcus pyogenes >gpIX69324ISPFCP.A_1 virR gene product [Streptococcus pyogenes] Length — 415 —  Score — 38 Identities Query: Sbjct:  Sbjct:  Sbjct:  (17.6 bits), Expect — 1.Oe+02, Poisson P(2) 10/26 (38%), Positives — 16/26 (61%)  1.0  —  —  77 RHLIEKSVRKIVSLLAASEAEVEQLL 102 R +K ++I LAA+EAE++L 111 RESSDKYKQEIGQLKAAAEAEAQKAL 136  Score = 37 Identities Query:  1.0  40 TPSPDVPLTIMKRKLMNTNDLE 61 TS VLT++ LNTD++ 19 TASVAVALTVLGTGLANTTDVK 40  Score — 38 Identities Query:  (17.6 bits), Expect — 1.Oe+02, Poisson P(2) 9/22 (40%), Positives — 13/22 (59%)  —  (17.1 bits), Expect 2.3, Poisson P(3) 9/29 (31%), Positives 15/29 (51%)  0.90  —  81 EKSVRKIVSLLAASEAEVEQLLSERAPLT 109 +++ +SLA AE+EL +A T 178 QQELAAVKSQLEAKNAEIEDLKQQDASKT 206  >gpIXO6826IATVIRB3 Agrobacterium tumefaciens virB operon [Agrobacterium tumefaciens] Length — 217  (plasmid pTil5955)  I  99  Page  14 Score — 61 Identities  Query: Sbjct:  Query: Sbjct:  (28.2 bits), Expect — 2.6, p 0.93 18/73 (24%), Positives — 32/73 (43%)  —  17 QFQGMKRKASSPVPLPPVTHLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQRHLDA 76 +F + + + RH + V S PL I T ++ Q T+ ++ LDA 7 EFDHVARHCAPSVATSTLAAIAKVESRFDPLAIHDNTTGETLHWQDHTQATQVVRHRLDA 66 77 RHLIEKSVR1(IVS 89 RH ++ + +1 S 67 RHSLDVGLMQINS 79  >5pIPO535OIVIB1AGRT9 VIRB1 PROTEIN PRECURSOR. >pirISOO777IB1AG55 virBi protein precursor Agrobacterium tumefaciens plasmid pTi15955 >pirIA28621IB1AGA6 virBi protein Agrobacterium tumefaciens p].asmid pTiA6 >gp)XO6826tATVIRB_1 Agrobacterium tumefaciens virB operon (plasmid pTi15955) [Agrobacterium tumefaciens] Length — 239 -  —  Score — 61 Identities Query: Sbj ct:  Query: Sbjct:  (28.2 bits), Expect 2.7, P — 0.93 18/73 (24%), Positives — 32/73 (43%)  —  17 QFQGMKRKASSPVPLPPVTHLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEI QRHLDA 76 +F + RH + V + + S ++ Q T+ ++ LDA PL I T 29 EFDHVARHCAPSVATSTLAAIAKVESRFDPLAIHDNTTGETLHWQDHTQATQVVRHRLDA 88 77 RHLIEKSVRKIVS 89 RH ++ + +1 S 89 RHSLDVGLMQINS 10].  >spP32532RRPP_PI1HE RNA POLYMERASE ALPHA SUBUNIT (EC 2.7.7.48) (NUCLEOCAPSID PHOSPHOPROTEIN). >pirIE4O234IRRNZ83 polymerase—associated nucleocapsid phosphoprotein parainfluenza virus type 1 (strain CI—14/83) >gpM74080IPIFCPHCY1A_2 phosphoprotein [Human parainfluenza virus type 1] >gpIM74O8OIPIFCPHCY1A_2 phosphoprotein [Human parainfluenza virus type 1] Length — 568 —  Score — 47 Identities Query:  Sbjct:  58 NDI.EESRQLTEEIQRHLDARHLIEK 82 N +EE+R L ++IQ +D+ + K 384 NKVEENRTLLKQIQEEIDSSRDLHK 408  Score — 44 Identities  Query: Sbjct:  (21.7 bits), Expect 3.le+02, P 1.0 9/25 (36%), Positives 16/25 (64%)  =  (20.3 bits), Expect— 3.0, Poisson P(2) 8/38 (21%), Positives — 20/38 (52%)  —  0.95  —  34 VTHLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQ 71 +T + PSP++ +++RK ++ ++ +E Q 208 ITDVITNPSPELEEAVLQRKKRRPTTIKRNQTRSERTQ 245  >gpIM73O45IDOGSNVD17A_1 Dog inserted sequence in spleen necrosis virus vector provirus clone. (Canis familiaris] Length — 163 Score — 59 Identities  (27.3 bits), Expect — 4.6, P 0.99 13/40 (32%), Positives — 20/40 (50%)  —  Page  15 Query: Sbjct:  41 PSPDVPLTIMKRKLNNTNDLEESRQLTEEIQRHLDARHLI 80 PSP +P + K L+ + + EE+QR L D H + 70 PSPSLPSSTEKSHLVPLMDARINAYIEEEVQRRLQDLHRV 109  >gpID25278IHUMORFO_1 ORE Length — 598 Score — 49 Identities Query: Sbj Ct:  (22.6 bits), Expect 1.6e+02, P 15/55 (27%), Positives — 24/55  —  Sbjct:  (17.6 bits), Expect 1.6e+02, Poisson P(2) 16/60 (26%), Positives 24/60 (40%)  —  1.0  —  23 RKASSPVPLPPVTHLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQRHLDAP.HLIEK 82 + RKA + L TPSP + T K L+ +E + L + R L+ K 204 RKALYSILDEVIFKLFSTPSPVIRSTATKLLLLMAESHQEILILLRQSTCYKGLPRLLSK 263  Score — 37 Identities Query:  1.0 (43%)  —  65 QLTEEI QRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAPLTGHSCYPEALL 119 +L+A + R L E++ + VEQL+ Q E +Q + PE L 485 QAQERLQHYFMGPALEERAQQHREALIAQI STNVEQLMXAPSLKEAEGKEPELFL 539  Score — 38 Identities Query:  [Homo sapiens]  (17.1 bits), Expect — 5.0, Poisson P(3) 9/33 (27%), Positives — 16/33 (48%)  0.99  21  KPKASSPVPLPPVTHLDLTPSPDVPLTIMKRK 53 +P MK + +P +VP+++KK L 1 MKPTGTDPRILSIAAEVAKSPEQNVPVILLKLK 33  Sbjct:  >gpIS66910IS66910_1 BRG1 [Homo sapiens] Length — 1613 Score — 43 Identities Query:  Sbjct: Query:  Sbjct:  Sbjct:  69 EIQRHLDALIEKSVRKIVSLLAASEAEVEQL 101 + R LD ++++ ++ + +A E+E L 355 QKPRGLDPVEILQEREYRLQARIA]4RIQELENL 387  Sbjct:  (18.9 bits), Expect — 63., Poisson P(2) 10/30 (33%), Positives — 16/30 (53%)  —  1.0  —  1.0  —  74 LDRHLIEKSVRKIVSLLAASEAEVEQLLS 103 +DARH+IE+++ S+A LS 700 VDARHIIENAKQDVDDEYGVSQALARGLQS 729  Score — 39 Identities Query:  1.0 (41%)  —  9 TI STMKVMQFQGMKRKASSPVPLPPVTHLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTE 68 T + K++ Q + P R + +P +PP + p M ++SR 295 TSTPQKLIPPQPTGRPSPAPPAVPPAASPVPQTQSPGQPAQPAPMVPLHQKQSRITPI 354  Score — 41 Identities Query:  (19.9 bits), Expect — 1.2e+03, P 19/93 (20%), Positives = 39/93  (18.0 bits), Expect 5.6, Poisson P(3) 8/22 (36%), Positives — 14/22 (63%)  —  122 RTHCFNWHSPTYEYALRHLYVL 143 R+ FN TYEY +++ ++L 847 RSGKFNVLLTTYEYIIKNKHIL 868  >pirIS289].61S28916 dystrophin  —  mouse >gpM68859IMtJSDYSA_1 dystrophin major  Page  16 muscle isoform [Mus musculus) Length — 3678 Score — 47 Identities Query: Sbjct:  Sbjct:  1.0 (59%)  —  64 RQLTEEIQRHLDARHLIEKSVRKIVSLLAASE 95 + L EI+ H D H ++++ +KI+ L S+ 2732 QDLQGEIETHTDIYHNLDENGQKILRSLEGSD 2763  Score — 46 Identities  Query:  (21.7 bits), Expect — 3.2e+02, P 19/32 10/32 (31%), Positives  =  5.7, Poisson P(2) (21.2 bits), Expect 8/17 (47%), Positives — 13/17 (76%)  —  1.0  —  60 LEESRQLTEEIQRHLDA 76 LEES+ + +E++ HL A 1473 LEESKMILDEVKMHLPA 1489  >gpM82829IHUMOPFD_1 fusion protein Length 744 Score — 59 Identities  [Homo sapiens]  1.0 6.1, P (27.3 bits), Expect 14/47 (29%), Positives — 22/47 (46%)  —  61 EESRQLTEEI QRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAP 107 + V L + AE E+L+ ER P + +T+ +Q ER A 153 EELDANTQALQEQDSAFGAVHTQMHAAVGQLGRARAETEELIRERVP 199  Query: Sbjct:  >spIP08799IMYS2 DICDI MYOSIN II HEAVY CHAIN. >pir1A266551A26655 myosin heavy slime mold (Dictyostelium discoideum) >gpIM14628IDDIMYRC1 chain D.discoideuzn myosin heavy chain gene, complete cds. [Dictyostelium discoideum] Length — 2116 -  Score = 51 Identities  (23.6 bits), Expect — 87., P — 1.0 14/47 (29%), Positives — 23/47 (48%)  —  62 ESRQLTEE IQRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAPL 108 R L E+++ + AR +EKS + + S L A E +L E+ 1067 EYTELNEKFNSEVTARSNVEKSKKTLESQLVAVNNELDEEKKNRDAL 1113  Query:  Sbjct:  45 Score Identities  (20.8 bits), Expect = 6.2, Poisson P(2) 10/26 (38%), Positives — 15/26 (57%)  —  1.0  —  1.0  =  61 EESRQLTEEIQRHLDARHLIEKSVRK 86 R+ +EK +K E+ R L EE+Q 926 EKVRDLEEELQEEQKLRNTLEKLKKK 951  Query: Sbjct:  Score — 39 (18.0 bits), Expect — 9.7, Poisson P(3) 13/17 (76%) Identities — 6/17 (35%), Positives 59 DLEESRQLTEEIQRHLD 75 D E+S++ T+ + +HL+ 715 DAEDSQKATDAVLKHLN 731  Query: Sbjct:  >gpIZ21513IRNINMEGLA_1 integral membrane glycoprotein [Rattus norvegicus] Length — 1199 Score  —  59  (27.3 bits), Expect  =  6.2, P  =  1.0  102  Page  17 Identities Query:  —  15/51  (29%), Positives  —  26/51  (50%)  27 SPVPLPPVTHLDLTPSPDVPLT IMKKLMNTNDLEESRQLTEEIQRHLDAR 77 SP+P ++ ++ R + EE Q HLD + +PS + P K ++N 260 SPMPEQILSTTLSSPSSNAPDPCAKETVLNALKEKKKRTVAEEDQLHLDGQ 310  Sbjct:  >gpIXO7278IDMLAMDMO_1 Drosophila mRNA for nuclear lamin Dm0 melanogaster] Length — 622 Score — 44 Identities Query:  [Drosophila  (20.3 bits), Expect — 8.2e+02, P — 1.0 9/22 (40%), Positives 11/22 (50%)  =  25 ASSPVPLPPVTHLDLTPSPDVP 46 ++PPPPTH SP P 25 SAGPQPPPPSTHSQTASSPLSP 46  Sbjct:  Score = 43 Identities Query:  (19.9 bits), Expect — 6.3, Poisson P(2) 12/54 (22%), Positives 24/54 (44%)  —  1.0  55 MNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAPL 108 +N + RQL + +RH L+EK + ++ + E + L+ + L 339 LNARIRDLERQLDNDRERHGQEIDLLEKELIRLREEMTQQLKEYQDLMDIKVSL 392  Sbj ct:  >5pIQO592OIPYC MOUSE PYRUVATE CARBOXYLASE (EC 6.4.1.1) (PYRtIVIC CARBOXYLASE) (PCB). >gpILO9192IMUSMPYR_l pyruvate carboxylase [Mus musculus] Length — 1178 Score — 48 Identities Query:  (22.2 bits), Expect 2.3e+02, P — 1.0 9/26 (34%), Positives 16/26 (61%)  —  16 MQFQGMKRKASSPVPLPPVTHLDLTP 41 ++ G++R +S+PV P V L+ P 11 LRLLGVRRSSSAPVASPNVRRLEYKP 36  Sbjct:  Score — 44 Identities Query:  (20.3 bits), Expect — 6.5, Poisson P(2) 7/15 (46%), Positives — 10/15 (66%)  =  1.0  127 NWHSPTYEYALRHLY 141 NW T++ A+R LY 606 NWGGATFDVAMRFLY 620  Sbjct:  >spIP29415ICX56_CHICK GAP JUNCTION CX56 PROTEIN (CONNEXIN 56) Length — 509 Score — 58 Identities Query:  (26.8 bits), Expect — 8.2, P — 1.0 12/39 (30%), Positives = 19/39 (48%)  —  10 I STMXVMQFQGMKRKASSPVPLPPVTHLDLTPSPDVPLT 48 ++T+ + S P P PPV P+P +P T G + 265 VTTLTPVMVTGESKPVSLPPPAPPVVVTTTAPAPVLPDT 303  Sbjct:  >pir1A453381A45338 connexin—56 [Gallus gallus) Length — 510 Score  (CX56).  =  58  chicken >gpILO2838ICHXCX56_1 connexin 56  —  (26.8 bits), Expect  —  8.2, P  =  1.0  — 103  Page  18 Identities  Query: Sbjct:  —  12/39  (30%), Positives  —  19/39  (48%)  10 ISTMKVMQFQGMKRKASSPVPLPPVTHLDLTPSPDVPLT 48 ++T+ + G + S P P PPV P+P +P T 266 VTTLTPVMVTGESKPVSLPPPAPPVVVTTTAPAPVLPDT 304  >pirID6OllOID6OllO repetitive protein antigen 3 Length — 259 Score = 47 Identities  Query: Sbj Ct:  Sbjct:  Trypanosoma cruzi  (fragment)  1.0 (52%)  56 NTNDLEESRQLTEEIQRHLDARHLIEKSVRI(IVSLLAASEAEVEQL 101 N L++ + L EE++R++ R IE ++ E++ R +L 91 NERLLDDKKCLEEELERNVLERERIESECRSRELVVGGLESKSREL 136  Score 41 Identities Query:  (21.7 bits), Expect 2.8e+02, P 12/46 (26%), Positives — 24/46  —  —  (18.9 bits), Expect — 8.3, Poisson P(2) 7/15 (46%), Positives = 8/15 (53%)  1.0  —  118 LLHFRTHCFNWHSPT 132 L+ RHC W PT 186 LVFLRAHCELWTDPT 200  Parameters: E 10., S 58 (26.8 bits), E2 W 3, T — 11 (5.1 bits), X — 22 H — BLOSUN62 H — 1, V — 100, B — 50 —gapdecayrate 0.5 (the default)  0.13, S2 (10.2 bits)  35  Statistics: Lambda — 0.320 flats/unit score, Lambda/ln2 0.462 bits/unit score K 0.134, H 0.576 bits/position Expected/Observed high score 62 (28.6 bits) / 123 (56.8 bits) * of letters in query: 168 * of neighborhood words in query: 4218 4 of exact words scoring below T: 0 Database: Non—redundant PDB+SwissProt+SPupdate+PIR+GenPept+GPupdate, 5:02 AM EST Mar 31, 1994 * of letters in database: 32,098,275 * of word hits against database: 10,602,499 • of failed hit extensions: 8,322,374 * of excluded hits: 2,278,900 * of successful extensions: 1225 I of overlapping HSPs discarded: 1144 * of HSPs reportable: 81 I of sequences in database: 113,405 * of database sequences with at least one HSP: 43 No. of states in DFA: 555 (55 KB) Total size of DFA: 101 KB (128 KB) Time to generate neighborhood: 0.02u 0.OOs 0.02t Real: 00:00:00 No. of processors used: 12 Time to search database: 73.44u 2.98s 76.42t Real: 00:00:15 Total cpu time: 73.52u 3.04s 76.56t Real: 00:00:16  104  

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