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Characterization of an 11s legumin-like storage protein gene from the gymnosperm picea glauca Márquez García, Magdalena Ivonne 1994

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CHARACTERIZATION OF AN uS LEGUMIN-LIKE STORAGE PROTEIN GENE FROM THE GYMNOSPERM PICEA GLAUCA by  MAGDALENA IVONNE MARQUEZ GARCIA B.Sc. (BioI.), National Autonomous University of Mexico (UNAM). 1987 A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES GENETICS We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1994 © Magdalena Ivonne Márquez-GarcIa,1 994  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 by the of head department my or by or his 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 British Columbia Vancouver, Canada Date  L  Abstract  The  amino  proteins  acid  in  sequence  all  seed  they  evolved  suggest  that  storage  protein  genes  angiosperms,  however  organization  of  This  the  first  gene.  A  is  protein legumin  gene  organization  have  report  2  the  exons  and  of  first  angiosperms.  is  available.  containing  a  long  Picea  characterized. to  be  this  and  introns.  The  similar  number  of  is highly conserved,  as  cloning  a  of  the  to  introns  it  is  98.7%  identical  to  is a  legumin  genes  of  509  in The  artifact  is  amino  previously  characterized legumin cDNA from Picea glauca. comparisons  The  however the position of  deletion being  is  uS  The nucleotide sequence contains  The deduced amino acid sequence  discussed.  storage  A deletion was found in the third exon.  possibility of  acids  structural  clone  found  in  the  seed  was  Seed  studied  gymnosperm  gene  introns  three  gymnosperms,  a  differs from those in angiosperrns, the  storage  ancestor.  gymnosperrns  and  four  seed  extensively  isolated  angiosperm legumin genes. five  common  regarding  in  genomic  a  been  data  of  including  from  genes  was of  plants,  no  the  homologies  lmino acid  Picea  and  other  species showed the presence of highly conserved sequences. Putative  regulatory  flanking  sequence  comparisons  sequences of  between  were  the  Picea  Picea  uS  uS  found  in  legumin  legumin  the  5’  gene  by  promoter  and  angiosperm SSP promoters.  12.  Table of Contents Abstract  ii  List of Tables  v  List of figures  vi  Acknowledgment  1  INTRODUCTION  2  LITERATURE REVIEW  2.1.  1  SEED STORAGE PROTEIN CHARACTERISTICS AND CLASSIFICATION  4  2.2.  GLOBULIN PROTEINS IN GYMNOSPERMS  5  2.3.  GLOBULIN STRUCTURE  8  2.4.  DOMAIN ORGANIZATION  2.5.  SYNTHESIS AND DEPOSITION OF SEED STORAGE  10  PROTEINS DURING ANGIOSPERM AN]) GYMNOSPERM DEVELOPMENT 2  .  6.  2.6.1.  11  GENE REGULATION  15  ABA REGULATION  16  2.7.  SSP ARE MEMBERS OF MULTIGENE FAMILIES  18  2.8.  GENE STRUCTURE  19  2.9.  REGULATORY SEQUENCES  20  2.10.  TISSUE SPECIFICITY AND TEMPORAL REGULATION  20  2.11.  THE ROLE OF CIS-ACTING ELEMENTS AND  CONSERVED  MOTIFS ON THE REGULATION OF LEGtJMIN GENE EXPRESSION 2.12.  3  DNA-BINDING PROTEINS  ..  22 24  MATERIAL AND METHODS  3 .1.  uS LEGtJMIN GENOMIC DNA ISOLATION  28  3.2.  RANDOM LABELING  28  3.3.  ?-GENOMIC DNA CHARACTERIZATION  29  3.4.  2k-DNA PREPARATION  30  111  3.5.  EXTRACTION OF  X-DNA  3.6.  CsC1 DNA PURIFICATION  32  3.7.  SOUTHERN BLOT  33  3.8.  MAPPING THE ?—GENOMIC DNA  33  3.9.  PLASMID DNA CLONING  34  3 .10.  VECTOR PREPARATION  35  3.11.  LIGATION OF VECTOR AND INSERT DNA  35  3.12.  COMPETENT CELLS PREPARATION  36  3.13.  ISOLATION OF PLASMID DNA BY ALKALI METHOD  37  3.14.  GENERATION OF UNIDIRECTIONAL DELETION CONSTRUCTS  .  FOR SEQUENCING  31  37  3.15.  SEQUENCING METHODOLOGY  39  3.16.  SEQUENCING GELS AND ELECTROPHORESIS  40  3.17.  4  PRIMER EXTENSION  40  RESULTS IDENTIFICATION OF A GENOMIC CLONE CONTAINING THE  4.1  uS LEGUMIN GENE  42  4.2.  uS LEGUMIN CODING REGION  4.3.  STRUCTURAL ORGANIZATION OF THE PICEA uS LEGUMIN  49  GENE 4.4.  53 PICEA GLAUCA uS LEGUMIN AMINO ACID SEQUENCE  4.4.1.  THE PICEA GLAUCA LEGUMIN PROMOTER REGION  4.5.1.  57  AMINO ACID COMPARISONS REVEAL CONSERVATION OF  HIGHLY CONSERVED SEQUENCES 4.5.  ....  PUTATIVE REGULATORY SEQUENCES  62 67 67  5  DISCUSSION  74  6  REFERENCES  82  iv  List of Tables  Table 4.1  Amino acid composition of the Picea 11S  legumin protein Table 4.2.  Percentage of amino acid identity among  legumin proteins: a) b)  61  Picea glauca versus gyrnnosperms;  Picea versus dicots and monocots  Table 4.3  Putative regulatory sequences  glauca uS legumin promoter  66 in the Picea 73  ‘7  List of figures  Figure 1.1.  Pathway for synthesis and processing of 11 S  seed storage globulins  14  Figure 4.1. Restriction enzyme digests and southern analysis of two k-clones  (1) XI5H-1 and (2)  XI5H-2  containing the uS legumin gene from Picea glauca  44  Figure  4.2. Restriction digests and southern analysis of  ? genomic clone containing a Picea glauca uS legumin gene  46  Figure 4.3.  Restriction map of the  -genomic spruce  clone XI5H—1 Figure 4.4  48 Nucleotide sequence of genomic DNA clones  S3.7 and E2.8 from Picea glauca uS legumin storage protein,  and  deduced amino acid sequence  50  Figure 4.5 Comparison of uS legumin genes from Picea glauca and  angiosperm subfamilies A and B  Figure 4.6. Comparison  of  54  uS legumin intron flanking  sequences  55  Figure 4.7. Deduced amino acid sequence of spruce uS legumin  60  Figure 4.8. Amino acid alignment of uS legumin proteins from Picea,  Pseudotsuga,  Pinus strobus,  cotton  (goshi),  .vi  oat(orysa),  Arabic7opsis,  pea,  sunflower  (helianthinin) 63  Figure 4.9. Determination of the transcription start site (+1) Figure 4.10.  70 Location of putative regulatory sequences  on the promoter of Picea uS legumin gene  72  vii  Acknowledgments  After  years  three  of  work  those whom I owe thanks. I  could  not  have  financial  assurance of  Canada Award Program,  it  difficult  is  to  undertaken  these  support.  thank The Government  I  studies  The CONACyT awards  and  for  friends  and  without  Program,  of  and for  I am perhaps most  in debt by BC Research for financial support, facilities  all  So I will begin at the beginning.  the loan received from Banco de Mexico. of  list  for the use  colleagues.  Special  thanks to Ben who offered me his supervision and big help, to  Craig who  taught me  as  much as  I  could  take.  To  John  Carison, my academic supervisor, whom I thank for accepting me as student,  and specially for the help at the end of my  thesis. I would like to thank professors, knowledge,  even  though  I  had hard  time  for sharing their at  the beginning,  due to my English difficulties. Very brothers  special  and  thanks  sisters,  to  whose  accompanied me to the end.  parents,  my  and  care  To Ian,  to  moral  all  my  support  who at the end of this  journey brought so much happiness to my life. To Stephanie,  Melody and Sheila,  and language teachings.  To my “Latin Family”  Victor and Lety, Nilda, Celia, and  Jorge  Oscar.  CH.,  Ivan,  for their cultural  Oliva,  Jaime, Ricardo,  Specially for their care,  Lynn,  in Vancouver:  Trini,  Gloria,  Jorge M.  Andrea  and for their  and  financial  support when I needed most.  viii  Chapter 1  INTRODUCTION  storage  Seed  constituent of  proteins  are  (SSP)  angiosperm and  an  gymnosperm  seeds,  the spores of more distantly related ferns. nutrients processes  for  the  necessary  germination for  the  (Shotwell and Larkins,  been  extensively  studied  in  and of  They provide  post-germination  propagation  Seeds contain 10 to 50% protein, protein  and  important  of  the  species.  most of which is storage These proteins have  1989).  angiosperms,  due  to  their  economic importance, but not many studies have concentrated on gymnosperm storage proteins. Even though angiosperms and gymnosperms ago,  the  diverged  function  and  same in both groups. the  maturation  from  (1986)  embryos  another  characteristics  330 of  million SSP  process  of  (Flinn  spruce  of SSP et  have proposed that  depends on the extent of  remain  somatic  embryos  zygotic  biochemical and  somatic  the  al,  1991b).  Redenbaugh  the  quality of  somatic  et al, embryos  storage protein accumulation.  markers  the  is similar to that of the  has been proposed that conifer storage proteins useful  years  It has been demonstrated that during  pattern of accumulation zygotic  one  for  developmental  embryogenesis  (Flinn  et  It  represent  studies al,  in  1991b,  et  Flinn  al,  sequences dicots. also  1993).  have  been  some  et al,  (Newton  the  highly  recent  Three  share  At  of  amino  conserved  papers these  1992,  acid  have  of  et al,  Hager  some  SSP  monocots  and  among  shown  regions  level  that  gymnosperms  conserved  1992,  Leal  sequence  and Misra,  However,  no data regarding SSP gene regulation have  been published.  To date only two gymnosperm cDNA sequences  1993).  been  have  et  (Newton  seeds recent  papers  conifers 1993;  published,  are  acid  al,  of  1992;  them Leal  that  suggested  at  belonging and  the  et  Flinn  al, some  sequences  Nevertheless,  These  1993). important  and  mRNA  nothing  is  (Leal  known  in  gymnosperms  regarding  and  about  SSPs  Two  and Misra,  on  information  conifer  1993).  level,  studies  stability  mRNA  to  Misra,  transcriptionally regulated  provided  have  both  amino  transcription.  the  structure  and  regulation of these genes in gymnosperms. Plant synthesis These  seed  storage  proteins  and accumulation  characteristics make  study gene  expression  contrast the  and  developmentally  them an  ideal  regulated.  model  to  the  relatedness  between  regulation  angiosperms  and  to  system  structure and organization of the genes, gene  in particular,  their  and  of  the genes  abundant  compare  mechanisms  and,  is  are  the  evolutionary  and gymnosperms.  encoding these proteins  However  in gymnosperms have not  been isolated and their structural organization is unknown. Characterization determine:  Whether  of the  these  genes  SSP genes  would  are  allow  one  to  structurally similar  2  (exons\introns)  between  angiosperms  and  gymnosperms,  and  cis-acting regulatory elements are similar in  whether the both groups. The  development  libraries  and  a  of  high  interior spruce at B.C.  cDNA  quality  libraries,  genomic  embryogenesis  system  DNA for  Research provide an opportunity to  study gene regulation during embryo development in spruce. The  contributions  characterization of sequencing  of  of  this  study  include:  coding  region  and  comparison  structure of the gene to other legumin genes.  protein promoter  2)  of  the  Deduction  sequence and comparison to other legumin  sequences. of  The  the white spruce uS legumin gene by  the  of the amino acid  1)  the  3)  Cloning  gene  and  and  sequencing  identification  of  of  the  putative  regulatory elements that could be important in the temporal and spatial regulation of the gene. To date this information  on  is the first report that provides direct a  complete  SSP  gene  in  gymnosperms.  The  sequence of the spruce uS legumin gene and the comparisons with homologous genes in angiosperms, about  structure  and  gene  provides information  organization.  data regarding putative elements  It  also  provides  that may play a role  in  the regulation of the gene.  3  Chapter 2 LITERATURE REVIEW  SEED  2.1  STORAGE  PROTEIN  CHARACTERISTICS  AND  CLASSIFICATION proteins  Storage albumins, Larkins,  are  classified  These  (albumins),  salt  (glutelins)  or  1985)  into:  (Shotwell and  due to their distinguishable physiochemical  1989)  prolamins  seeds  globulins, glutelins and prolamines  characteristics.  Black,  from  SSP  (globulins),  The  or  proteins  Cereal  in  alkali  (prolamines)  predominant  glutelins.  and  solubility  acid  alcohol  aqueous  .  show  in  storage  water  solutions  (Bewley  and  cereals  are  proteins  occur  predominantly in the endosperm and are limiting in lysine. In  dicot  the  seeds  most  limiting  in  globulin group,  storage  proteins  are  which occur in the cotyledons and  globulins and albumins are  abundant  methionine  and  cysteine.  Within  two major forms of salt soluble  the  proteins  are resolved from one another on the basis of sedimentation characteristics, llS  and  7S.  Both  and fall are  into two different  insoluble  at  pH  4.7  size classes  in  O.2M NaC1.  Because globulin proteins have been best characterized in legumes,  the  llS and the 7S  legumins and vicilins,  fractions  are referred to as  respectively. However,  other trivial  names derived from the genus of the plant are also given. The legumin fraction has a sedimentation constant of 11-13S and molecular weight  of  36Ok1D,  composed of  six  identical 4  subunits  of  60  glycosylated,  KD.  The  have  two  legumin  subunits,  components:  subunit of 4OKD and the basic,  or  subunits are covalently linked by vicilin  The  l8OkD.  weight of a,  f3  and  a’  fraction has  a  7-9  1  the  which  are  acidic,  not  or  subunit of 2OKd.  a  These  a single disulfide bond. value,  S  and  a molecular  It is made up of three major subunits  of 76KD,  72 KD and 53KD.  2.2 GLOBULIN PROTEINS IN GYMNOSPERMS Studies based on solubility characteristics in conifer seed have shown the presence of globulin, as  crystalloid protein and albumin type proteins,  et al,  1991a;  and Green,  Stabel et al,  Picea  abies  accumulation mature  1990; Hakrnan et al,  1990; Green et al, (1990)  Stabel et al, in  also referred to  1991).  somatic  storage  embryogenesis.  proteins  of  42,  and degradation upon onset  embryos  Hakrnan  et  al,  protein  of  281W,  (1990) and  1990; Misra  described 3 major storage proteins  during  of  (Flinn  described showed  that  an  They  33  and  22K]D  of  germination.  additional  mature  found in  storage  embryos  contain  more storage proteins than immature embryos. This indicates that,  in  as  storage  angiosperms,  proteins  maturation. have  been  1988)  and  in  synthesis  gymnosperms  and occurs  accumulation during  of  embryo  Storage proteins with similar molecular weights described Picea  in  glauca.  several These  Pinus  species  findings  suggest  (Gif ford, that  seed  storage proteins may be conserved among conifers. 5  interior  Comparing  stages Flinn eC al, 33,  24,  23  and  mature  spruce  storage  and  somatic  different  found by SDS-PAGE  (1991a)  KD  from  SSP  proteins  zygotic  that the 41,  accumulated  embryos.  embryo  These  only  in  proteins  correspond to the storage proteins found in protein bodies isolated from mature seed embryos amount  these  of  influence  of  storage  ABA  (see  of  proteins  ABA  interior spruce.  are  moderated  regulation).  Misra  by  and  The the  Green  have shown that in the mature seed of white spruce,  (1990)  70% of the total protein content correspond to crystalloid proteins, studies classes  the major storage proteins shown  have  such as  (Gifford,  similar  Pinus,  results  Norway  1988; Misra and Green,  stages  (41KD), (Flinn  accumulates  accumulate et  later  at  al,  during  (35,  early  different  Douglas  of  33,  fir,  seed etc.,  24 and 22KD)  different  at  Albumin-like  protein  similar to legumes,  embryo  and  developmental  cotyledon maturation,  the rest of the storage proteins, accumulating  Other  1991)  199lb).  stages  in  spruce,  Interior spruce globulins albumins  (35 kd range).  maturation.  By  and  start two  dimensional electrophoresis storage proteins appeared to be composed of various isoforms analysis (1991a) pattern  under found  a  55-57  KDa  1991a).  conditions, protein.  The  Flinn  By PAGE et  al,  characteristic  of storage proteins under reducing conditions was  composed of 33, 55-57KD,  non-reducing  (Flinn et al,  24 and 22KD proteins,  suggesting  that  disulfide  but no proteins with  linkages  exist between  6  the 33 and 24 and 23 KDa proteins, analogous to legumins in angiosperms. (1992) have shown that the SSP content in  Allona et al,  differs  pinaster  Pinus  represent  glutelins  from  70%  of  other  total  conifers,  protein  in  that  content  while  globulins and albumins constitute 26% and 4%, respectively. In this study the authors compared the structure and amino acid sequence of the glutelin protein to other plants and these glutelins  concluded that  are homologous  to  the  uS  There are two basic differences between the P.  legumins.  glutelins  pinaster  and  the  uS  extraction  requires  alkali  glutelins)  and  the  b)  legumin  solution  basic  proteins:  to  (similar  character  of  the  a)  the  rice larger  subunit which appears to be acidic in the rest of the uS proteins. These data agree with the results from Jensen and Lixue  where within 31 species of Pinaceae studied,  (1991),  all except the 12 Abies species were shown to contain ilS legumins.  The  amino  acid sequence (Allona  pinaster between  species  lack  liS  legumins  but  have  glutelin like proteins. There is no data regarding  instead, the  Abies  them.  et  Jensen  from Abies to  al,  1992)  and  or  Lixue  to  compare with Pinus define  (1991)  the  suggest  homology that  the  absence of legurnin type proteins in Abies species may have something  to  these seeds,  do  with  the  shorter period  of  viability of  compare to Picea or Pinus.  Legumin-like proteins in seeds of Gingko biloba have been reported  (Jensen  and  Berthold,  1989).  A  50  KD,  dimer 7  separates into 28  and 21 KD  subunits,  disulfide bonds. The molecular weight, properties  and  characteristics  protein  been  has  heterogeneity  and  (Templeman  DeMaggio,  storage proteins, reported  by  that  the charge,  correspond  reported  demonstrated  that are linked by  for  the  Onclea  contain  et  al,  (1987)  for  The fern Osmunda cinnamomea,  1990).  The  similarities  share  fact  of  globulin  that  between  conservation  strong  also  sensibilis  both  globulin  all  seed  plant  storage  Matteuccia  also contains  globulin storage proteins of 5.5S and 11.3S deMaggio,  It  7S and uS, which are comparable to those  Templemann  struthiopteris.  leguinin-like  angiosperms.  fern  1990),  to  subunits  (Templeman and  storage groups  proteins  proteins  suggest during  a the  evolution of seed plants.  2.3 GLOBULIN STRUCTURE Globulins have been extensively studied in both cereal and dicot  seeds.  In  cereals  component whereas in as  80%  of  the  shown  been  and  ,  Picea  1991b;  globulins  (Gifford, spruce  species Roberts  1988;  an  important  In gymnosperms,  seed protein. and  component of gymnosperm seeds 1990)  not  most dicots they account for as much  total  that  they are  [Picea abies  (Gifford,  et  Allona  albumins  al, et  (Misra and Green,  1988;  1990;) al,  1991),  the  major et al,  (Stabel  Flinn et al,  several  1992),  are  it has  Pinus  Douglas  Gingko biloba  fir,  1991a  species Norway  (Jensen and  8  Berthold,  and Fern species  1989),  (Templemann and deMaggio,  1990)]. vicilin  The  legume  various from  seeds  salt  dilute  molecular  polypeptides  (Nielsen,  extracts  weights  are  1989)  of  around  best  characterized They  .  seed  meal  KD  and  180  are  as  from  isolated  trimers  contain  with  random  combinations of non identical subunits. Each trimer has one or  N-linked  two  transcripts  glycosyl  from  translationally  the  and  groups.  7S  The  genes  are  weight species  gene  modified  post-translationally.  emanate from preproteins of 70 KD that, signal peptide,  primary  The  co  proteins  after loosing their  are cleaved to produce the high molecular and a smaller polypeptide of 20 KID.  (51 KD)  Legumin polypeptides have also been best characterized in  legume  (Nielsen  particularly  seeds,  et  al,  1989)  sunflower uS protein (1980)  concluded  subunits  in  on  soybean  electron  and  pea  microscopy  of  (helianthin), Richelet and co-workers  that  arranged  Based  .  from  each two  complex  trimers  is  composed  et  (Nielsen  al,  of  six  1989)  Similar results were found for rape seed uS globulin using x-ray  scattering  molecular  (Plietz  weight  of  360  et KD.  al,  1983).  Subunits  The in  hexamer has  the hexamer are  not glycosylated and need not all be identical. forms  of  the  llS  families present polypeptides with  a  basic  subunits  in several  components, isoelectric  are  part  species.  of  a  the  Different multimeinber  Each subunit has  two  one with an acidic and the other point.  The  two  components  are  9  linked  by  a  single  (Nielsen,  soybean  disulfide  bond.  Legumin  subunits  can be separated into  1986)  two groups.  Subunits in group I have uniform apparent molecular and contain more sulfur than members of group-Il. in  the  same  group  are  88%  to  90%  The differences between the observed at gene level  weight Subunits  homologous,  homology among members of different groups  is  in  however  40%  to  50%.  different members can also be  (see chapter  2.8.).  2.4 DOMAIN ORGANIZATION A relationship of predicted domain organization between 7S and liS globulins has been proposed, and bean  physical (Argos  which differs  for  characteristics  et  al,  1985).  Domain  based on amino acid  soybean, I  is  pea the  and 2 NH  significantly between 7S and uS.  french  terminus Domain II  contains common regions and domain III is the COOH-terminus half and is highly conserved.  Nielsen  (1986)  proposed that  the hydrophobic and most highly conserved domain is domain III.  Argos  et  al,  (1985)  proposed  that  the  single  disulfide bond between domain I and III play an important role  in  maintenance  of  conformation  of  the  subunit.  Evolution of a common precursor for the vicilin and legumin families  has  comparisons  been (Gibbs  proposed based  et  al,  on  amino  1989).  The  acid  presence  hypervariable regions between domains II and III, for  the  size  differences  between  the  two  sequence of  accounts  globulins.  The  insertions within these regions vary in length and consist 10  largely of repeated aspartate and glutamate residues, acidic  very  and  are  predicted  to  exist  in  a  are  helical  conformation (for review see Shotwell and Larkins 1989). By amino  comparing has  acid  shown  been  sequences  that  there  from different  are  repeats  of  8  species to  38  it  amino  acids corresponding to the hypervariable region at the end domain  of  polar,  II.  These repeats acidic  mainly  characteristics inserts  are  vary  can  residues. common  a  in  contain a high proportion of  length,  Although  structural  amino  acid  these  feature,  the  composition  and  location within and between species.  SYNTHESIS AND DEPOSITION OF SEED STORAGE PROTEINS  2.5  DURING ANGIOSPERM AND GYMNOSPERM DEVELOPMENT The synthesis of storage protein in seeds is regulated  during the  development.  end of  In  the mitotic phase  seed maturation when with  the  at  (Muntz,  of  1989).  Chrispeels,  unknown.  Seeds  protein  storage  and  at  takes  starts the  at  end of  place.  protein  endoplasmic  rough  Along  formation,  reticulum  takes  Seed storage proteins are synthesized  and  polysomes  transferred  to protein bodies 1985).  expression  and finishes  cytoplasmic  1979)  and Black,  gene  desiccation  of  the  membrane-bound  synthesis  seed  increase  proliferation place  general  from  (Nielsen  et  (Bollini their  al,  site  1989;  and of  Bewley  The mechanism of protein sorting remains contain  each  has  more a  than  one  class  characteristic  of  storage temporal 11  accumulation  pattern.  Despite  the  differences  angiosperm and gymnosperm embryo development,  synthesis and  deposition into storage organs is quite similar Storage  globulins  polysomes signal  are  synthesized  precursor  as  sequence.  by  polypeptides  The  signal  between  (Fig 2.1).  membrane-bound  with  2 NH  peptide  terminal  directs  the  translocation of the nascent polypeptide into the lumen of the  endoplasmic  removed.  reticulum  after  Soon  are  precursors  and  translation  assembled  is is  into  co-translationally  complete  the  trimers  within  globulin the  endoplasmic reticulum and then transported to vacuoles via apparatus.  Golgi are  into  cleaved  remain  linked  process,  Once  the  accumulation  the vacuole,  acidic  and  disulfide  by 115  type  subdivide  Vacuoles  in  of  basic  bonds.  trimers form  to  storage  the  uS  precursors  polypeptides After  the  assemble protein  proteins.  which  proteolytic  into  hexamers.  bodies  for  the  Double-labeling  of  storage proteins of pea has shown that some protein bodies contain  both  7S  and  115  Millerd,  1981). Microscopic analysis of protein bodies from  nearly  mature  embryos  globulin  of  proteins  Interior  (Craig  spruce  and  (Picea  glauca/Picea englemanii) showed that both globulin proteins were present in the same organelles Protein  bodies  are  confined  triploid endosperm cells storage  seed  homologous  to  tissue the  in  in  (Flinn et al,  to  the  angiosperms. gymnosperms  protothallium  l991b).  cotyledon In is  or  the  contrast  the  haploid  and  heterosporic  ferns.  It 12  develops independently and before fertilization of the egg cell  (Jensen and Bethold,  1989).  Protein bodies have been  identified in mature and near mature seeds and reported  rarely  have  been  at very immature stages in angiosperms or  gymnosperms. Many  storage  during  modifications correct  size  products  of  proteins  deposition  to  1989)  The  (Muntz, the  undergo  legumin  genes  translational modifications. hydrophobic  component  .  post-translational convert primary  undergo  A signal  co-  the  to  the  translation and  sequence  removed during  is  them  post-  that has  synthesis  a of  the precursors, while cleavage to form the acidic and basic probably  polypeptides angiosperms,  cleavage  occurs has  been  in  protein  reported  bodies.  always  to  In  occur  between an aspargine and a glycine, with the later becoming the N-terminal data  on  biloba has  of the basic polypeptide.  legumin-like shown  that  protein there  terminus of the basic subunit  is  of  the  a Asn  However,  recent  gymnosperm  Gingko  residue  (Hager et al,  at  the  N  1992).  13  Subunit Structure 1.  Synthesis  Oligomer Composition  Intracelular Compartment  of  I  preproglobulin 2. Removal of signal peptide  I  I  I I  3. Disulfide bond formation  RER  ri  4.!stassemblyinto 8S trimers  J 5 i r  5. Transport to protein body via Golgi  1  6. Proteolic processing  Golgi  I  Protein Bodies  7. 2nd assembly into 11 S hexamers  Fig 2.1  Pathway for synthesis and processing of uS seed  storage globulins. (In the and  the  figures black  Taken from Shotwell and Larkins,  1989.  the white areas represent the oc subunits, areas  the  j3  subunits.  RER  =  Rough  endoplasmic reticulum)  14  2.6. GENE REGULATION encoding  Genes of  subject  storage  intensive studies  expression.  gene  seed  Seed  proteins  towards  storage  have  been  the  the understanding of  proteins  are  encoded  by  a  diverse gene set that is highly regulated during the plant life  cycle  genes  et  (Goldberg  expressed  are  developmental  al,  tissue  under  regulation  Seed  1989).  and  storage protein  specificity  therefore  are  an  system to study the control of gene expression. to  interactions  which  tissues  between  the  embryo  excellent The extent  and  surrounding  regulate development remains uncertain as  signals that form the basis of these interactions. attempts  many  to  elucidate  performed in angiosperms, The  isolation  corresponding  and cDNA5  these  and  processes  are  the  However  have  been  especially in the past 10 years.  characterization encoding  of  seed  mRNA5  storage  and  their  proteins  have  produced a vast amount of information regarding amino acid sequences,  number  of  temporal  genes,  regulation,  and in many cases data about  the  themselves.  genes  concerning  published  There the  genes  is  not  for  and  the structure of much  SSP5  in  information gymnosperms.  The cDNA sequence for the vicilin gene of spruce al,  1992),  spruce gene  in  cDNA sequence  (Newton, Douglas  (Newton et  for legumin and albumin genes  in preparation), fir  spatial  (Misra  and  of  c]DNA sequence for legumin Leal,  1993)  have  led  to  interesting comparisons between angiosperms and gyrnnosperms at this level which permit speculation about the evolution 15  of these proteins. However, no data from genomic DNA clones has  been  published,  therefore  the  organization  of  these  genes in gymnosperms still remains unknown.  2.6.1 ABA REGULATION the  Although  regulation  of  storage  influenced by the developmental  stage of  the details  of how this  occurs  is  al,  Information  from  1991).  protein the  is  seed/embryo,  not known  phytohormone  genes  (Bauinlein  action  et  during  developmental events has provided a better understanding of embryo  development.  phytohormone  absicic  acid  important physiological et al, the  via  processes  receptors Evidence  obscure.  a  in plants  number  transduction  at  embryo  the  physiological  (Redenbaugh et al, et al,  (199lb)  of 1986).  maturation  and  level  suppression  1988).  of  spruce  accumulation  of  storage  premature  gymnosperm  germination.  in  Roberts et al,  somatic proteins Globulin  embryos and  in the  maturation  (1990) ABA  of  It has also  embryo  have shown that including  maturation  of  pathways  been demonstrated that ABA plays an important role regulation  of  that ABA plays a major role  precocious germination (Mundy and Chua,  proper  the  (Finkelstein  and/or  in  of  mediates  that  The mode of action  indicates  control  shown  1988).  monocots and dicots the  been  (ABA)  1985; Mundy and Chua,  hormone  remains  has  It  and Flinn  during  the  results  in  suppression  of  proteins  including  16  legumin and vicilin as well as albumins are accumulated in response to ABA. been  has  ABA  accumulation  found  to  regulate  embryogenesis  during  at  storage  the  transcriptional  level in seeds of diverse species of angiosperrris  et  al,  Mundy  1987;  increases precocious mRNA5 al,  immature  in  and in  1985)  Chua,  the  in  (Finkeistein  of  case  ABA  storage protein  (Roberts et al,  gymnosperms that  seed  angiosperms  in  (Kuhiemeir  Exogenous  1988).  accumulation of  embryos  demonstrated  been  and  protein  et  It has  1990). legumin,  both  protein and mRNA accumulate in response to ABA at specific (Finkeistein et al,  developmental time in angiosperms and gymnosperms  increase  produced an mRNA,  (Roberts et al,  suggesting  1990).  Sorbitol treatments  in ABA that preceded storage protein  that  osmotically  induced  ABA  stimulates  storage protein expression in rapeseed (Wilen et al, Interior  spruce  accumulate  zygotic  legumin,  1990).  and somatic embryos have shown to and  vicilin  mRNAs,  from the cotyledon stage  stage,  in  presence  the  1985)  albumin to  protein  late embryo maturation  (Flinn  ABA  of  storage  et  al,  1993)  The  amount of proteins accumulated and the transcript levels of these  storage  proteins  in  somatic  concentration dependent  (Roberts  of  storage  1993). excised  Stimulation zygotic  demonstrated osmotic  embryos  (Finkeistein  stress  ABA  by  et  levels  embryos  et al,  osmotic  in  ABA  1990; Flinn et al,  protein  al,  were  1985).  accumulation  in  stress  has  In  response  vegetative  tissues  been to are  17  (Skriver  increased  that  suggested  and  osmotic  Mundy, effects  increased ABA  and  1990), on  embryo  it  via  proteins  accumulated in broad bean cotyledons  to high osmoticum (18% sucrose). the  effects  of ABA can be  (Bostock  rice  have  (1993)  and  demonstrated  protein  and  somatic  embryos.  storage  Recently  osmotic  stress  transcript  combined  the  caused  synthesis  of  These  angiosperms  (Bostock and Quatrano,  have an ABA pathway  in response  Flinn  et  al  storage  fluoridon  proteins  that  1992),  in  in (an  and high osmotic  storage  suggest  stress  accumulation of  effect  inhibited.  results  legumin  induced  inhibitor of endogenous ABA biosynthesis) treatment  and  are  It has been suggested that  1992).  protein  The  Vicilin  triggered by osmotic  Quatrano,  been  development  mediated  levels.  has  to  similarly  gymnosperrns  be to may  that is induced by stress.  2.7 SSP ARE MEMBERS OF MULTIGENE FAMILIES Like  other  eukaryotic  genomes,  characteristic  in plants.  Genes  proteins families from  a  (vicilins (Ellis few  to  et as  1988;  many  belong  et  Heim  as  families  encoding globulin  legumins)  and  al,  multigene  20  al,  members.  to 1989)  are  storage  multigene ,  varying  Hybridization  experiments and cDNA sequence analysis have confirmed that legumin multigene families are divided into two subfamilies A and B 1993;  (Baumlein,  1986;  Shotwell and Larkins,  Depigny-This,  et al,  Dure  III,  1989;  1988;  Turner,  Breen and Crouch,  1992; Wang et al,  et al, 1992;  1987; Takaiwa et al,  18  1991; acid  Shotwell  al  et  1990;  identity between  however  between  the  Pang two  members  et  al,  subfamilies  of  the  also confirmed for some species (Pich and Schubert,  Domoney et al, De  The  .  is  same  percentage of identity is about 80%.  families  1988)  40  amino  to  50%,  subfamily  the  RFLP experiments have  the presence of multigene  1993; Domoney and Casey  1985;  1986)  Pace  et  al  have  (1991)  shown  by  in  situ  hybridization that genes encoding the 2 legurnin subfamilies in  Vicia  encoding shortest  Eaba  are  arranged  in  clusters:  the  genes  legumin A are located in the long arm of the two subtelocentric  chromosome  is in a less terminal position; are  two  located  in  submetacentric  the  pair.  pairs  et  centromere  those coding for legumin B  non-satellited Casey  whose  al  arm  of  the  have  (1988)  longer  also  shown  that the two legumin genes for Pisum sativum are located in two different chromosome pairs.  2.8 GENE STRUCTURE Genes  for  uS  legumin subunits  share  common  features.  The coding region is approximately 2.7 Kb including 2 or 3 introns subfamily  (Shrisat  et  and  A,  B  al,  1989;  respectively.  variable sizes,  70 bp to 600 bp,  well  conserved  for  rape  (Baumlein  et  Goldberg,  1989).  soybean, al,  Nielsen  1986;  The  al,  1989)  in  are  of  introns  however the positions are  broad bean, Rodin  In angiosperms  et  et  pea,  al,  and  1992;  the position of  oilseed Sims  and  intronsi  19  and 2  of  introns  the 2  subfamily B, 3  and  from  correspond to  the positions  subfamily A genes  (Baumlein  of al,  et  All the intron/exon junctions follow the GT/AG rule  1986).  for eukaryotes.  2.9 REGULATORY SEQUENCES Recently,  attention has been given to the study of the  flanking sequences and to the structural and functional  5’  analysis of the upstream region that regulates seed storage  tobacco,  Petunia  revealed  an  This  processes.  regulation of  of  plants,  transgenic  ArabicIopsis,  to  investigate  storage protein  seed  evolutionary  as  control  gene  expression  of  regulatory  conservation  tissue  includes  such  specificity  and  temporal  the genes as well as correct regulation and  for  processing  use  and  regulating  sequences has  The  genes.  protein  mRNAs  and  cleavage and glycosylation)  proteins  (transient  (Bustos et al,  signal  1991).  2.10 TISSUE SPECIFICITY AND TEMPORAL REGULATION Fusion experiments of globulin genes to reporter genes and  the  subsequent  introduction  into  transgenic  plants  have demonstrated that SSPs can be expressed in the correct size and composition only in mature seeds. (1989)  used a  T-DNA construct  containing  Shrisat 3.4  et  Kb pea  al, LegA  fragment fused to a nos reporter gene which was introduced into  tobacco  plants  that  the  Kb  3.4  via  fragment  Agrobacterium.  contains  all  They of  the  demonstrated information  20  for  necessary  specificity and correct processing  seed  the primary transcript and the legumin precursor. al,  showed that a 1.2  (1988)  of  Ellis et  Kb upstream sequence of  the  pea LegA gene was able to direct synthesis of the legumin protein  transgenic  in  deletion  by  showed  in  expression  tobacco. analysis  transgenic  legumin protein was  of  et  only present  in  (1989)  al,  and  transient  that  transgenic  LegA,  plants,  tobacco  Baumlein et al,  leaf tissues.  Shrisat  seeds  and  absent  in  have cloned a 4.7 Kb  (1991)  fragment of the LegB from Vicia faba containing the coding 2.4kb upstream and 0.3  region, was  functional  after  transfer  only  expressed  and was  plants,  of  analysis  Kb 3’,  legumin genes  fragments  of in  (pGV18O)  were  LegB  front  of  into in  have  for high levels of expression.  and showed that transgenic  seed  defined  it  tobacco  tissue.  Deletion  important  regions  Partially deleted promoter  inserted  in  a  vector  plasmid  the nptll gene and transferred into  tobacco via Agrobacterium. Expression was detected by nptll and in situ hybridization to an antisense  enzyme activity, RNA LegA  probe. gene  The  (Ellis  et  sequence  flanking  The  expression. further  results  upstream  is  revealed  that  similar  1988),  about  1.2Kb  al  enough  possibility was  to of  suggested.  confer minor A  the  to of  high  positive  construct  the levels  pea LegB  of  elements containing  only 0.2Kb of the upstream sequence resulted in a dramatic reduction of  nptll activity.  that  5’  a  97  bp  fragment  Shirsat et al  of pea  LegA which  (1989)  showed  contains  the 21  CART  and  expression. the  However  flanking  5’  boxes  TATA  the  to  increased by  suggesting that  involved.  from these results:  sufficient  synthesis  sequence,  elements must be  not  was  induce  increasing  additional  cis  An interesting question arose  What are the DNA sequences involved in  the temporal and spatial regulation of these proteins?  To  address this question different approaches have been used: sequence analysis of legumin promoter regions to define  a)  Cis-acting  b)  sequences;  DNA-motifs  in vitro mutagenesis  c)mobility  and;  shift  assays  of to  specific test  the  binding of nuclear factors or known transcription factors.  THE ROLE OF CIS-ACTING ELEMENTS AND  2.11  CONSERVED  MOTIFS ON THE REGULATION OF LEGUMIN GENE EXPRESSION In  the  regulation  search of  gene  putative  several  sequences  these  for  specific  expression  sequences  motifs in  have  seed  been  transcriptional  in  conserved  to date al,  (Riggs et at,  1989;  These  also  1991)  .  found.  are  of in all  Shirsat et al, referred  to  and  A 549 bp 5’  the  Legumin  proteins  The  role has  28  could  of  been  legumes  bp, studied  RY  repeats  direct  for  (Dickinson et al,  flanking sequence containing CART, Box  the  1990; Ericson et  as  storage protein genes other than legumin 1988).  in  The legumin box is a  sequence  TCCATAGCCATGCAAGCTGCAGATGTC present  storage  regulation  studied and in some cases confirmed. highly  involved  legumin  TATA  synthesis,  suggesting the involvement of the legumin box in regulation  22  of  gene  expression  et  (Shirsat  al,  1989).  This  was  also  suggested by the absence of expression when using a 97 bp 5’  (as mentioned above) which only contained 12 bp  sequence  of the legumin box. only  legumin  in  Since the leguinin box  genes  (Riggs et al,  genes  but  1989;  in  all  is present  seed  storage  Chamberland et al,  not  protein it has  1992),  been suggested that presumably this sequence has a role in the  of  regulation  elucidate  tissue  function  the  Baumlein  performed. reduction  of  containing  et  specificity. of  this  al  (1991)  expression when  using  legumin box,  arguing  the  Many  attempts  to  sequence  have  been  observed  a  fold  a  200  bp  that  10  5’  sequence  the presence  of  the legumin box within this sequence plays no role in the high  level  expression  of  possibility that elements  is  not  clear.  of  is  dependent  However,  expression  gene  comparing  the  full  glycinin  Gy2  expression  promoter  without  observed  a  present,  function  developing  seeds.  The  on other  Cis  studies  have  other  that the legumin box plays an important role as  suggested enhancer  its  in  (Lelievre  of  a  promoter  ten-fold  suggesting  construct from  leg-box,  the  reduction that  the  al,  et  Lelievre the  leg-box  .  containing  soybean  when  1992)  or et  the al  the same  (1992)  element  was  has  role  a  By  not in  regulating the amount of expression of the gene. Chamberland et  al  (1992)  have  shown  that  the  legumin  box plays an important role in —conglycinin transcription. In  the  case  of  soybean  -conglycinin gene  there  are  two 23  well  defined  legumin  resulted in a  boxes  and  the  mutation  of  both  ten-fold reduction in the transcription of  the gene. Three other regulatory elements closely related to the sequences  consensus  in  TG(T/A/C)AAA(G/A) (G/T) the  -1203  1989). of  and  -549  glutelin  genes  were reported in 5’  flanking  in  pea  region  cereals  legA between et  (Shirsat  al,  This sequence has been implicated in the expression protein  storage  genes  by  nuclear  DNA-binding  protein  experiments as well as by promoter analysis.  2.12 DNA-BINDING PROTEINS An  important  linking  stimulus  step  primarily  regulation  to  sequences  accumulated  Evidence  of  signal to  transduction pathway  alterations  of  eukaryotic  the binding of nuclear proteins,  factors  on  the  perception  gene expression is trans-acting  in  specific 5’  to  to  date  Cis-elements  located  gene  region.  the  coding  indicates  transcription  i.e.,  in  that  a  sensitive  cell-type  or  developmentally specific manner is achieved by multiplicity of  interactions  between  promoter  enhancer  sequences  and  trans-acting factors with either stimulatory or repressive ( Meakin and  functions  Cis-acting expression glutenin, lectin,  have barley  Gatehouse,  elements been  controlling  identified  hordein,  conglycinin  1991)  and  oliseed french  in  seed-specific  maize rape  bean  zein,  napin, phaseolin  wheat soybean genes 24  (Jordano  al,  et  and  1989,  references  therein)  Conserved  .  elements have been postulated to play an important role in activation of gene  transcription by the binding of  trans  acting nuclear proteins. Examining sequence specific DNA-protein interactions, by DNA-protein binding and mobility shift et  (1990)  al,  assays,  demonstrated that nuclear proteins  Shirsat strongly  bound the -549bp flanking sequence. However a truncated  -  124bp LegA construct fragment containing the complete legbox  sequence  with  6  5’  additional  did  bases  not  bind  nuclear proteins. footprinting  DNA  experiments  demonstrated  interaction  of a nuclear protein from pea seed (LABF1) with the -549 to fragment  -316  Gatehouse,  of  1991)  LegA .  5’  flanking  region assays  retardation  Gel  (Meankin  and  showed  the  specific interaction between two LegA promoter fragments 540  -316  to  proteins. +40  did  protein.  and  -833  The promoter not  form  to  sequence  stable  Developmental  and  -584)  of  seed  nuclear  LegA between  complexes  regulation  pea  and  with  -316  seed  tissue  (-  to  nuclear  specificity  between nuclear proteins and legA promoter was demonstrated by gel retardation assays.  The nuclear protein binding the  region showed a molecular weight  promoter  of  84  -  116  KD  that was determined by elution and renaturation of protein from  PAGE-SDS.  confirmed (1991)  by  showed  Its  function  competition the  tissue  as  assays.  DNA-binding Meankin  specificity  and  protein  and  was  Gatehouse  developmental  25  regulation  this  of  binding  factor.  Extracts  from  peas  during development were tested with a probe consisting of 549 to -316 LegA promoter. extracts  12  to  19  days  The factor was detected in seed  after  anthesis  (DAA).  The  seed extract interacted strongly to the probe. extracts was  recognized the probe. specific  seed  and  The  that  its  binding  suggested  1989),  transcriptional  that  enhancer.  it  Since  that  DAA  LABF1  activity  was  (Thompson and  may  a  15  No pea leaf  evidence  temporally correlated with synthesis of mRNA Larkins,  -  act  low  as  a  level  of  transcription occurs when LABF1 protein is not detectable, transcriptional  enhancement  rather  than  induction  was  suggested. In  studies  Jordano  et al  on  sunflower helianthinin gene  (1989)  detected nuclear  an AlT rich region upstream of Binding  competition  expression,  proteins  that bind  the helianthinin promoter.  experiments  showed  that  sunflower  embryonic and somatic nuclear proteins bound to the french bean phaseolin gene and to the carrot DcG3 embryo specific genes,  suggesting  between showed site,  plant  that  binding  species.  that  the  fused  to  In  sequence, a  activities  the  same  containing  reporter  gene  truncated CaMV 35S promoter,  are  report the  (GUS)  conserved  the  authors  protein  binding  and  driven  enhanced expression  in  by  a  seeds  in transgenic tobacco plants. Elements are  mostly  that AlT  bind  rich,  nuclear and  do  proteins not  show  in  other any  species  particular  26  e.g.  sequence conservation,  et  al,  Pha  1989),  vulgaris  phaseolus  in  lectin  soybean  1989),  sunflower helianthinin  et  (Jofuku  al,  (Kitamura et al,  soybean globulin genes  (Jordano  (Riggs 1987),  1990;  et  al,  and  two  Itoh et al,  1993) its  Despite binding  to  the  et  have  sequence, been  Shirsat et al,  (1989)  al,  conserved  legumin box 1991;  and Gatehouse, Riggs  highly  suggested  regulation by soluble proteins,  detected  1990;  that  no  as  proteins (Meankin  Itoh et al,1993). an  alternative  to  the CATGCATG motif may form  a Z-DNA structure in vivo. One possibility is that after an activator protein binds 316), that  to the upstream region  (-549  to  -  the CATGCATG motif may adopt an altered conformation enhances  transcriptional  the  recognition  complexes.  Itoh  for  et  or  al  of  passage (1993),  also  suggested that assuming that the leg box could be a binding site  for  nuclear  factors,  these  they may require  unstable,  or  different  site  for  other  factors factors  may  be  binding  interaction with these motifs  as  very at  a  found  in yeast mating type regulatory proteins.  27  Chapter 3 MATERIAL AND METHODS  uS LEGUMIN GENOMIC DNA ISOLATION  3.1.  spruce  The  uS  legumin cDNA XI5H was  Craig Newton at B.C.  Dr.  Research.  obtained  The cDNA was labeled and  used as a probe to isolate the white spruce genomic clone. k-library from a  from  uS legumin  —  The EMBL3 Eastern white spruce total genomic constructed  was  partial  by  L.  Sau3a digest,  DeVerno  DNA was  (PFNI).  Isolated  cloned into a BamHl  site.  3.2.  RANDOM LABELING 20  ng  of  heated at 100°C DNA.  XI5H  cDNA  for 5 mm.  or  E2.8  DNA  in  11111  H20  were  to separate the double stranded  After heating the DNA sample was placed on ice and all  the labeling components were added (2J.ti lOX labeling buffer, 2J11 dNTPS jil  (2 inN each G,  0.1 M DTT,  T,  C),  2J11 & P-dATP 2  1J.Ll pN6,  (5000  ijil BSA (1 mg/ml),  Ci/inmol;  Dupont),  1  1 unit  of Klenow enzyme  (BRL). The labeling reaction was allowed to  proceed at  temperature  twice  room  precipitated  overnight.  probe was  then  with half volume of 7.5 M NH OAc 4  and  2.5 volumes of cold 95% ethanol, as  carrier  12,000  rpm,  temperature  (-80  C; 0  15  mm.,  30  mm.).  4°C,  The  The  using of tRNA (2 mg/ml) Sample pellet  was was  and resuspended in 100 jil water.  centrifuged dried  at  at  room  lj.Ll of sample  28  was  used  verify  to  P-ATP 32  incorporation  in  a  liquid  scintillation counter.  3.3. ?-GENOMIC DNA CHARACTERIZATION Three ? clones Dr. Craig Newton were ?-phage dilutions  XI5H-3)  used to characterize the genomic DNA.  10 mM MgSO ) 4  were mixed with 0.1 ml of  ER1647 bacteria host and incubated at 37°C tubes agar and  for 20 mm. (10  plated  (O.45J1m) phage  on  TB  4°C  placed at  Following the  tryptone,  g/l  provided by  (lO-lO plaques /ml of SM ( 50 mM Tris-HC1  100 mM NaC1,  pH 7.5,  XI5H—2,  (XI5H-l,  5  incubation 3  g/l NaC1,  plates,  6  incubated  overnight.  in 10 ml-falcon  g/l at  ml  of TB  agar)  37°C  were  for  7  was placed on top of each plate for 2 mm.  to  adsorb  to  filter.  Filters  were  denaturing  solution  (1.5  M  NaC1;  followed by neutralizing solution HC1 pH 7.5)  0.5  M  NaOH)  filter  off  the  soaked in  for  (1.5 M NaC1;  and  to allow  peeled  plates and placed DNA side up on 3M Whatman paper,  added  hr  One nylon hybridization  top  5  mm.  0.5 M Tris  Filters were air dried on  for another 5 mm..  Whatman paper for 20 mm. and exposed to UV light for 7 mm. to  denature  hybridization Pyrophosphate; hr  at  added, Filters  65°C, and were  DNA.  Filters  solution  were (5X  placed  SSPE;  in  1%  10  SLS;  ml  pre  0.1%  Na  200 Ig/ml denatured salmon sperm DNA) the  radiolabeled  hybridization washed  twice  cDNA  probe  proceeded with  2X  above)  (see  overnight  SSC  pH  7  for 2  at  (0.15M  was  65  C. 0  NaC1,  29  0.015M Na-citrate)  and 0.1%  air  temperature  dried  room  at  SDS  film with intensifying screen overnight  exposure  and  exposed  (-80 °C,  film  the  for 30 mm.  was  were  with  1  temperature. for  20  identified,  developed.  mm.  ml  bacteria  plaque forming units  and  X-ray  The  and  positive  to X-ray film.  for  2  culture  hr  in SM,  plated  incubation at 37  to  at  incubated  on  TB  9 io  pfu/ml of  overnight in SM media  the number of  C, 0  XI5H-1 and XI5H-2 were added toa of ER1647 culture growth  mixed by  ( 5 x 108 cell/ml ) containing 4 ml of  inversion  and placed at  37  C 0  for  Lysates were added to 250 ml TB media and shaken (37  C; 0  5 hr).  5 ml  250 ml of lysed culture of  each  DNase  I  and  RNase  were  added  addition  to  NaC1  1M  final  10 mm.;  4  C 0  ).  (RT),  to  25  lJig/ml  lysates  and  followed by the  concentration,  swirling and placed 1 hr on ice. rpm;  mm..  at 2000  and shaken at 37 °C 10 mm.  at room temperature  of  20  of chloroform were added to each  incubated 30 mm.  11,000  agar  (pfu/ml) was determined.  10 ml falcon tube containing 10 ml  rpm  room  2-DNA PREPARATION 5 x  SM,  Kodak  transferred  shaken  host  then  three single phage  of phage were made  After overnight  plates.  3.4.  each,  SM  Dilutions with  picked,  65°C  overnight). Following  Then by aligning filter to agar plates,  tubes  to  identified by aligning filters  plaques were  plaques  at  dissolved  by  Lysates were centrifuged g of  PEG  8000  (10%  final  30  concentration)  were  stirring  cooled  room  (RT),  overnight. were  pellets  added  to  supernatant,  dissolved  by  on iced water and placed in the cold  After  centrifuging  resuspended  in  8  (11,000  ml  SM  rpm;  10  media.  8  mm.) ml  of  chloroform were added and samples centrifuged  (3000 rpm;  mm.;  bacteriophage  4 °C),  collected by centrifugation  particles °C)  the aqueous phase recovered and  and resuspended in 0.5 ml SM  (25,000  rpm;  2 hr;  overnight;  (4 °C;  15  4  rocking  platform).  EXTRACTION OF 2-DNA  3.5.  The bacteriophage solution was gently resuspended in SM  media,  mg/mi), °C).  and  EDTA  25 jil SDS 10%  This  step was  (0.5  M  ),  proteinase  51i1  K  (5  were added and incubated 1 hr at 56  followed by two  chloroform:phenol  (1:1)  and one chloroform extractions. DNA in the aqueous phase was precipitated isopropanol  with  -20  at  centrifugation ethanol, pH 8.0,  7.5  M  overnight.  °C  rpm,  (14,000  1 inN EDTA) with  following on  volume  20  OAc 4 NH DNA  mm.),  the a  and CsC1 purified.  EcoRI  and  instructions  0.8%  agarose  gel  Hindlil from  and  was  1  with  %  sample was  restriction  in TEA buffer  70  by  (10 mM Tris-HC1  A 10 El].  supplier  volume  pelleted  washed  dried and resuspended in TE buffer  digested  run  half  enzymes  (Pharmacia), containing  and EtBr  (0.5 jig/mi).  31  CsCI DNA PURIFICATION  3.6.  and  AS-DNA  CsC1  purified. generation  deletion  the  acid pellets  purified  and  (E2.8  constructs,  S3.7) were  for  were  used  CsC1  for  southern  the blot  as well as for sequencing reactions.  After nucleic  DNA  plasmid DNA preparations  of  hybridization,  plasmid  DNA  extraction  resuspended  equilibrium  by  in  2-  step  2.4  ml  or  plasmid  TE buffer were  sedimentation  in  cesium  chioride-ethidium bromide  (CsC1-EtBr)  gradient.  4.2g of CsC1  and  EtBr  added  the  plasmid  JA-21  Beckman  400  jil  of  10  mg/mi  centrifuged  solution,  tubes  partially  was  chloride  solution  solution  placed with  filled  centrifuged Beckman tube  was  g/100  the  cesium  for  18  RT)  hr  with ml  bottom  8  of  a  from  light  the  tube.  DNA The  balanced,  rpm  light,  of  and  the  solution, (40,000  ml  TE),  ultracentrifuge rotor. protected  in  to  A Ti70.1 quick seal ultracentrifuge  filled  (63  at  rpm;  (6000  centrifuge for 10 mm..  were  20  ;  cesium plasmid  tube  sealed in  °C)  a  and Ti  the  and  the  the  position  of  to UV light.  The lower DNA band was removed from the tube using a  added  and  DNA  extracted  water  saturated  4  isobutanol.  times The  transferred to a 30 ml corex tube  70  After centrifugation,  plasmid band determined by exposing the tube  syringe with a wide bore needle.  was  Three volumes with lower  an  equal  aqueous  of  1 ml  TE were  volume phase  of was  and precipitated with 3  32  volumes of cold 95% centrifuged  was with  rpm;  (15,000  dried  ethanol,  cold  overnight at 20 °C. The sample  ethanol  30  mm.),  at  room  the pellet  rinsed  temperature,  and  resuspended in TE buffer.  SOUTHERN BLOT  3.7.  After digesting 5 DNA each, and  Sail,  Tris-base,  mM  (0.89  were  samples  Promega,  of  combinations  following  them, on  loaded inN  0.89  with EcoRI,  a  boric  acid,  gel  run for 4 hr at 75 V/H.  was washed in HC1 solution  (21.5 ml/l)  three  NaOH  10 mm.  times  three  times  in  JIM  20  containing 0.5 JIg/mi EtBr,  1M  from  conditions  agarose  1%  Hindill,  TBE  EDTA)  The gel  in 3M NaC1  30 mm.,  1M  QAc 4 NH  and blotted to a nitrocellulose  filter  for  10  denatured  mm.,  in  for 4 hr at room temperature. After blotting the filter was allowed  dry  to  at  room  and  temperature  DNA  fixed  5  mm.  under UV light. After this step the filter was hybridized to the  labeled  cDNA  probe  same  the  using  method  described  above.  3.8.  MAPPING THE -GENOMIC DNA In  DNA,  to  generate  a  map  the  of  legumin  genomic  restriction digestion of the k-DNA and plasmid DNA were  performed. DNA  order  were  In all cases CsC1 purified DNA was used. digested  with  each  combinations of them: Ec0RI,  of  SalI,  the  following  PstI,  BamHI,  5 g of  enzymes  or  Hindlil. All  33  were  enzymes  conditions  obtained  were  carried out  fragments  resulting  from  were  Promega  as  and  suggested by  separated  restriction supplier.  according  to  The  size  by  electrophoresis through a 0.8% agarose gel cast in 0.5x TBE, containing EtBr next  DNA  to  (0.5  samples  2k-DNA size markers were loaded  and  were  used  as  a  reference  to  determine sizes. After electrophoresis was completed the gel photographed under UV  light.  Sizing  the  different  DNA  fragments was done manually by measuring the distances  and  was  referring to the size markers, negative  the  scanner.  of  After  and also by computer scanning  photograph  the  pictures  were  through  taken  gels  a  Sparc  were  1  (Sun)  blotted  and  hybridized to cDNA probe as described above.  3.9.  PLASMID DNA CLONING pEMBL  The convenient cloning  and  cloning  sites,  with  the  compatible ends or  SalI  gels.  and  vectors  to  due  use  to  systems the  were  multiple and  Both DNA fragment and Vector DNA were same  restriction  for cloning.  the  expression  for deletion experiments  and usefulness  sequencing reactions. digested  pGEM-3Z  products  k-DNA was  were  An EcoRI 2.8KD fragment  enzyme,  produce  digested with EcoRI  visualized (E2.8)  to  in  0.8%  agarose  and a Sail fragment of  3.7 lCD (S3.7), were gel purified (using the prep-A-gene Kit, Promega),  and cloned in vector pEMBL  or pGEM-3Z vector  (SalI 3.7  (EcoRl 2.8 KD fragment)  ).  34  3.10. VECTOR PREPARATION or pGEM  pEMBL  vector  EcoRI or Sail as needed,  1  °C;  recircularization reaction was  of  (0.01 t/pmo1 ends in 100; phosphate  5’ the  vector  stopped by adding  2  groups  during of  Jil  with  chloroform:isoamyl  and  37  prevent  ligation.  0.5 M EDTA.  DNA was phenol/Chloroform extracted once, extracted  digested with  then treated with calf intestinal  (ClAP)  remove  to  hr)  were  JIg)  following the Promega instructions  to get complete digestion, alkaline phosphatase  (10  ClAP Vector  the aqueous phase  alcohol  (24:1),  and  DNA  precipitated with 0.5 volumes of 7.5 M ammonium acetate and 2 volumes of 95% ethanol collected by with  95%  centrifugation  ethanol,  concentration  C, (-80 0  was  dried  30 mm.). DNA pellets were  (12,000  and  determined  rpm;  resuspended by  10 in  washed  mm.), 0. 2 H  absorption  The  DNA  spectroscopy  260 A  3.11. LIGATION OF VECTOR AND INSERT DNA Vector DNA and insert DNA were mixed at  molar ratio in ligase mix Ligase, at  1 p1 10 mM ATP,  room  complete, cells  (1 jil ligase 5x buffer,  0 to 10 Jil) 2 H  temperature. plasmid DNA was  l;l and 1:3  After  1 unit DNA  for overnight reaction  ligation  transformed into  reactions SURETM  were  competent  (see competent cells below) Aliquots of  50 p1 of competent  cells were thawed on  35  ice  and 2.5Jil  of  the plasmid ligation reaction were  and  incubated  15  mm.  on  ice.  To  increase  transformation  efficiency a heat shock at 37 °C for 1 mm. followed by 2 mm. each  and  tube  on ice.  incubation at  Bacto-yeast extract, ainpicillin,  Ig/ml mg/ml)  for  14  -  10 16  was performed,  200J1l of LB medium were added to 37  Cells were plated on LB  mm..  added  °C was  allow  for  45  to  (10 g/l Bacto-tryptone,  60  5 g/l  5 g/l NaC1, pH 7) plates containing 50 Il  hr.  IPTG  and  (1M),  Recombinant  50  white  X-Gal  colonies  (20 were  selected and single bacteria colonies inoculated on 2 ml LB medium containing 50 .tg/m1 ampicillin,  incubated 8  14 hr.,  -  followed by miniprep plasmid DNA isolation procedures.  3.12. COMPETENT CELLS PREPARATION  5  g/l  lml YT/Mg  (20g/l bacto-tryptone,  NaC1  g/l  2.5  colony  (Stratagene) were grown  ) 4 MgSO and  was  grown  to  5 g/l yeast extract,  inoculated mid-log  phase.  then added to a lOOmi warm YT/Mg in 500 ml to  pelleted  600 A by  =0.6.  Bacteria  centrifugation  were  cells  (3,500  rpm,  gently resuspended in 40 ml of cold TfBI 2 MnCl  with  100 mM KC1,  suspension was cold TfBII  10 inN CaCl , 2  centrifuged as  (10 mM Na-MOPS pH 7.0,  15% glycerol),  Bacteria  on  ice,  C) 0 2  (30 mM KOAc,  15% glycerol). above,  mi  TM Sure  flask and  chilled 15  1  and 50 inN  The bacterial  resuspended in 5m1  75 mM CaC1 , 2  of  10 inN KC1,  aliquoted and frozen in liquid nitrogen and  stored at -70°C.  36  3.13. ISOLATION OF PLASMID DNA BY ALKALI METHOD DNA  Plasmid  isolated  was  by  procedure described by Maniatis et al  the  alkali  (1982).  lysis  1.5 ml of the  plasmid cultures were centriguged at 12,000 g for 2 mm. microfuge  The bacterial pellets were resuspended by  tubes.  vortexing in 100 111 10 inN EDTA,  ph 8.0,  ice cold lysis buffer 50 inN glucose),  freshly prepared solution  of  II  (Potasium acetate  inversion,  by  12,000  5  g  (0.2N NaOH,  The  mixing  1%SDS),  150 il of ice-cold  pH 4.8) were added and mixed  incubated 5 mm. mm..  (25 mM Tris-HC1,  followed by the addition  by inversion and incubating 2 mm. at RT. solution III  in  on  ice,  supernatant  and centrifuged at separated  was  and  one  volume of phenol:chlorophorm (1:1) was added, vortexed for 1 and centrifuged 5 mm.  mm.  phase was for  5  12,ooo  g  with 2.5  precipitated  mm.  at  10  RT.  mm.,  at 12,ooo g.  DNA was  The upper aqueous  volumes  pelleted by  washed with 70%  of  ethanol  (95%)  centrifugation at  ethanol,  vaccuum dried,  and resuspended in 50 p.1 TE buffer.  3.14. GENERATION OF UNIDIRECTIONAL DELETION CONSTRUCTS FOR SEQUENCING The  TM erase a-base-system  construction of  the  sequence  of  legurnin  containing  subclones gene  analysis.  and  The  (Promega)  progressive  promoter,  system  is  was used for  to  based  deletions  facilitate on  the  the  use  the of  37  exonuclease III to digest DNA from a 5’ end,  leaving  while  phosphotioate  4  a  filled  base  end  3’  protruding or blunt  protruding  intact.  The  end  digestion  or  an  a  produced  a  series of deletions of increasing size that were exposed to SI  which  nuclease  from  remainded  the  removed  the  Exo  digestion.  III  neutralized and heat inactivated. added  the  to  ligated Half  to  circularize  to  of  reaction  (Stratagene)  competent  colonies  each  of  deletion  were  preparations  cells.  used After  time  SI  blunt  deletion  was  stranded  tails  nuclease  was  Kienow DNA polyinerase was  generate  the  reaction  each  single  that  containing to  transform  selected  followed  were  plasmids. SUreTM  transformation 4  were  performed  ends,  and  by  10  to  plasmid  enzymatic  restriction to determine the samples to be sequenced. Two sequencing coding S3.7 the  CsCl-purified purposes,  region  and  the  0.7kb  of  DNA  inserts  E2.8  (containing  3’  flanking  were  used  the  for  complete  sequence)  and  the  (containing 1.4kb of the promoter sequence and 2.3Kb of coding  region).  Bacteria  containing  the  E2.8  or  S3.7  insert were grown overnight in two hundred and fifty ml of YT broth  (8 g/l bacto-tryptone,  g/l NaC1) incubation  5 g/l bacto-yeast extract,  5  with ampicillin added at 100 jig/ml. Following the at  37  0  nucleic  acids  were  isolated  using  the  alkaline lysis method.  38  3.15. SEQUENCING METHODOLOGY fmolTM  Promega  The  sequencing  sequence the uS legumin gene.  system  was  used  to  The fmol system uses Taq DNA  polymerase which is stable at 95°C and which replicates DNA and allows use of a thermocycling apparatus. (Twin  at 70 °C, block  system,  TM  ERICOMP).  specific  legumin  Forward,  100  , 2 MgC1  mM  polynuclotide 50  different  primers  (27mer 24mer  (5 -GCCTAGGCGTTAATTGTCATAGACGTA-3’),  2Omer Reverse)  10 pmoles ‘y-ATP,  Three  were end labeled  lOX T4 buffer  mM  kinase,  DTT,  1  30  mm.  (10 pmoles primer,  (500 mM Tris-HC1 ph7.5,  mM  spermidine) 37  C. 0  The  5  units  T4  kinase  was  inactivated at 90°C 2 mm. and the labeled primers were then used for sequencing proposes. with  4.5  jil  sequencing  of  10mM MgC1 ), 2  1.5il  TaqDNA polymerase of  the  l-2J11 template DNA were mixed  buffer  labeled primer, (5u/JIl).  (250  mM  Tris-HC1  0 2 H  to  18  111,  pH9.0, and  For each set of reactions  enzyme/primer/template  were  added  to  each 4 Ll ml  0.5  eppendorf  tube containing 1 jil of each of the four d/ddNTP  mixes  [40IM  dCTP,  (G  7-Deaza  60p,M ddGTP],  dTTP,  4OJiM dCTP,  dATP,  40p,M dTTP,  dGTP,  40pM dATP,  A  dGTP,  40J.LM dATP,  401M  [4OjiM 7-Deaza dGTP,  700J1M ddATPI, 4OEIM dCTP, 40J1M dTTP,  T  placed  in  the  thermal  401.LM  [40J..LM 7-Deaza dGTP,  401M  l200mnM ddTTPI, 40p,M dCTP,  cycler  40LM  4011M dATP,  C  [ 40pM 7-Deaza  400J1M ddCTP]).  drop of mineral oil was added to each tube, and  dTTP,  preheated  One  spun for 2 sec. at  95°C  for  2  39  The PCR program used for the sequencing reactions was  mm. as  follow:  95  (annealing),  °C  70  30  sec.  °C 1 mm.  (denaturation),  60  for 30  30  °C  sec.  cycles  total.  After reactions were completed 3 jil of stop solution  (10 mM  NaOH,  formamide,  95%  cyanole)  (extension)  0.05%  bromophenol  were added to each tube.  blue,  0.05%  xylene  Samples were heated for 2  at 70 °C just before loading on sequencing gels.  mm.  3.16. SEQUENCING GELS AND ELECTROPHORESIS 5 Urea,  ml  of  long  50%  6 ml lOx TBE,  ranger  solution  (J.T.Baker),  21  25 ml H 0 were mixed and filtered. 2  g  25 ji  1 TEMED and 250 p.1 of 10% ainmonium persulfate were added and the  solution  was  transferred  to  a  50-60  ml  syringe  injected in between the sequencing gel glass plates. gel  polymerization  loaded.  (1-2  Electrophoresis  hr)  sequencing  was  performed  After  reactions using  and  were  0.6X  TEE  running buffer at 30 watts for 3-6 hrs. Once electrophoresis was completed plates were separated and the gel transferred onto Whatman 3M paper, at  80°C  for  1  hr  covered with saran wrap, vacuum dried  and  exposed  to  a  Kodak  x-ray  film  overnight.  3.17 PRIMER EXTENSION mRNA  from  white  spruce  stages was obtained from Dr. proembryo  and mature  proembryoand  Dave Cyr  embryo mRNA5  mature  (BCResearch).  were  used per  embryo 3 p.g of  reaction.  40  Proernbry mRNA was used as a control and no RNA was used as a negative control. Three samples of mature mRNA one sample of proernbryo InRNA and no RNA sample were mixed each with  primer previously  (see primer  labelled  NaCl heated at 80 °C for 60 secs. of  at  55  o  for  mg/ml  BSA,  inhibitor,  250  enzyme) °C.  was  in  and immediately after each 42 °C,  55  O  15  mm.  All  samples  0.01 UN  added,  M  DTT,  dNTP5,  0.5  were  removed  and  M  u/ml  0.01  ,0.05 2 MgCl  units  Reverse  reactions were allowed  7.5 M NH Ac) 4  Transcriptase  for  1 hr.  at  42  (37 °C  ethanol  at -80 0 C for 20 mm.  Samples  and ran along with  using the same  RNase  vol.  were redissolve in sequencing running buffer methodology)  the  0.1 M Tris pH8.5,  followed by ethanol precipitation  95% and 1/2 vol.  and  (0.1 M KC1,  Reactions were stopped by the addition of RNase A  10 mm.)  0.3M  Pro embryo and no RNA mixtures were placed  Reverse Transcriptase mixture 0.1  labelling),  the mature embryo mRNAs were placed at  65 0 C for 15 mm.  10 ng of 27mer  S3.7  (2  (see sequencing  sequencing reactions  27mer primer.  41  Chapter 4  RESULTS  IDENTIFICATION OF A GENOMIC CLONE CONTAINING THE uS  4.1  LEGUMIN GENE The EMBL3 Eastern white spruce -genomic library was screened like) (C. 2,-3)  with  the  Newton,  spruce  XI5H  cDNA  probe  unpublished results).  legumin  (uS  Three plaques  (-1,-  that strongly hybridized to the probe were selected. A  partial  restriction  map  was  obtained  for  each  DNA  followed by a Southern blot hybridization using the of the X15H cDNA as a probe.  sample end  5’  Phages XI5H-2 and XI5H-3  show  the same restriction pattern and are therefore considered to be identical clones the  southern  (data not shown)  blot.  southern blot  of  Fig  4.1  shows  Only one was used for  .  restriction  2 XI5H-1 and -2.  clones  exhibited different restriction patterns. although  hybridized  the  probe,  suggesting  the  presence  leguinin  family  hybridized  in  the  Picea  probe,  of  at  very least  it  was  restriction  characterization.  A  constructed which  showed  that  weakly two  for  for  members XI5H-1  clones  ?  XI5H-2, of  further  X15H-1 an  (E2.8)  the  strongly  for  clone has  fragment  two  and  Nevertheless both  selected map  this  17.9Kb containing a 2.8 Kb EcoRI  The  Since  glauca.  digests  insert  was of  and a 3.7  42  Kb  Sal  I  fragment  (S3.7)  The  Three  fragments  from  sequencing vectors, in  a  flanking  XI5H-1  E2.8  pGEM-Z3  sequence  subcloning,  strongly hybridize  the  cDNA  2 XI5H-1 restriction map is shown in figure 4.3.  probe.  S3.7  that  was  vector  were  subcloned and  S4.7  subcloned  was  subcloned  in  in  a  in  E.coli  pEMBL  vector,  which pUC9  contained  vector.  3’  After  the 3 plasmid DNA samples were CsCl purified.  In  order to obtain a full length genomic DNA the E2.8 and S3.7 were and  selected. they were  Both  of  large  Nielsen eC al,  strongly hybridized the  enough to  legumin genes  based on  them  contain a complete  in angiosperms  uS  et al,  (Ellis  probe gene 1988;  These two fragments were sequenced by  1989).  the PCR method and only when the sequence was not clear the sequenase Kit DNA was  from USB was used for confirmation.  only partially sequenced.  approximately sequenced  from  0.3kb the  from 5’  the  end  to  length containing 18 bp of 5’  3’  E2.8 end.  the  SalI  has  a  The  The E2.8  Sail E2.8  site,  flanking region,  site DNA  2350  bp  clone  contains  1.4  Kb of promoter region,  was in  483 bp of 3’  non-coding region and an open reading frame of 1867 bp. S3.7  at  1867  The  bp àf  coding region and 483bp of 3’ non coding sequence.  43  I 0  0) CD  -.4  /0)  C,’  CA)  -‘‘.3  w  t  1%)  CA)  ttt  0  I  -  ‘.3  43  CD  -4  0)  C,’  CA)  enzyme digests 4.1. Restriction (A) and clones Southern hybridization (B) of 2 (1-5) XI5H-1 and (6-10) XI5H-2 presumably containing the us legumin gene from Picea glauca. 3.Lg of k-DNA were digested EcoRI (lane 1,2,6 and 7); HindIll (lane 3 ,5,8 and 10); EcoRl/Hindill (lane 4 and 9); separated in 0.8% agarose gels (A) in presence of EtBr, and (B) blotted on Hybond N and hybridized P-labeled 5 cDNA probe. Bordering lanes to the 32 markers. show DNA Fig  45  0’  -‘  g I,  I  S  0  -‘I 0) CD  0)  Cu  c)  Fig  4.2.  analysis  of  Restriction genomic  digests  clone  and  (XI5H-l)  Southern containing  Picea glauca us legumin gene. 2ig of ?-DNA were digested with either EcoRI (lane 1); EcoRl/Hindlil (lane 2), EcoRl/Sall (lane 3); EcoRI/PstI (lane 4); lane (Lane 6); HindIII/PstI HindIII( 5); Hindlil/Sall (lane 7); PstI (lane 8); SalI (lane 9); SalI/PstI (lane 10); and were separated in 0.896 and (B) agarose gels (A) in presence of EtBr, Hybond and hybridized the blotted on N to P32 labeled E2.8 probe. ?-DNA markers are shown.  47  SE  fII’  HH H  III  Coding Region  Promoter  450  SEBEEHS 11111  I  1867  S ‘IRAI  3’ sequence  I  I  483  Fig 4.3 Restriction map of the 2-genomic spruce XI5H-1. The clones that strongly hybridize the spruce us legumin cDNA are shown (E2.8 and 83.7). 83.7 and E2.8 contain a deletion 77 bp long, marked as a solid bar across them. The cDNA is 1738 bp in length and is shown in the figure for comparison. The empty bars inserted in the cDNA sequence correspond to the introns. The line at the bottom represents the sequence obtained from the three regions; numbers correspond to length in bp. Note that the restriction map do not correspond to the cDNA. LA= ? left arm; RA= right arm; E= EcoRl, 5= SalI, H= Hindlil, B=BamHI  48  uS LEGUMIN CODING REGION  4.2.  In  order  sequence  to  nested deletion  experiments  the using  complete the  coding  region,  Promega erase-a-base  system were performed. All deletion products were re-ligated ligation  and  competent  reactions  cells.  were  White  colonies were selected,  used  to  containing  SURETM  transform  inserts  over  blue  grown in YT media and DNA extracted  as detailed in materials and methods.  2 .Lg of DNA of each  sample were restricted with EcoRI and separated in agarose gels, was  to select samples to be sequenced.  compared  to  cDNA  the  sequence  All sequence data  using  NA-align  and  NA-  compare programs.  These comparisons showed a 98.7% homology  between  and  the  cDNA  gDNA,  with  the  exception  of  a  77bp  deletion. Figure 4.4 gives the complete sequence of the gene including 5’ the  and 3’  genomic  DNA  flanking sequences.  are  different  substitutions are present  from  10 nucleotides  the  cDNA.  5  of  in  these  in the third position of codons,  four of which do not produce a change in amino acid,  while  one  these  changes  a  substitutions  serine are  in  for  an  the  arginine.  second  Another  position  result in a change to a similar amino acid.  of  3  of  codons  and  One nucleotide  that differs from the cDNA in the first position resulted in a  change  changes and  from  are  an  found  therefore  do  arginine in any of not  to  a  cysteine.  None  of  these  the highly conserved regions,  greatly affect  amino  acid homologies  with other legumin genes.  49  AATATTAACA TTAAAAAT TTATGTAGGA ATATTTAAGC CAATAAAAA TATAAATATT  60  TAAGTAATAA AAAATAAAAA ATATAAAATT TAAGTAATAA ATTTTTTCCT CGTGGAACGT  120  ATTTTTTCCT CGTTAGATGT GAACACATAC ATTGACAGCA GCATTTCCTT AAACAAACAC  180  TCAACTTT ACACGTCGAA TCGTACGACA TTACACGACA CGCCGGAGAG TAGCCGCATC  240  ACACGTGATG AAGATTCCCT TTGGCCTTAA GCCCATGTGG CTCTCAGGAG TAGATATAGC +1 CTTAATCATA TCGCCCTTCG CATGCTATAA_AGCTAATAAT ATTCAACAAC AGCAGGGAC  300  CAGCCTGTGT ATAAAAACAC GAAGAAGCAT CTAGGAATTC AAAACGAAGC AAGAGAAATG  420  AAGGGGAAGA TGATGAGATC AGCGCGTTGT CCACTGATGC AGATACTGTT AATTGCCTCT H  =  Q  ILL  IA  S  360  480 8  GCCTGCTTTC TTTTTCTCTC CCTGTCACT GTATCACCTG TAACTGCAAT TTCCCAGCAA L L S T V S P V T A I A C F L F S S Q Q  540 28  AGJJGAGGAA GAGGTCGTCG TTACGATGAG CAATCATCGT CATGTCGGAG GCTGCGGCGG Y D E R R G R G R R S S S C R R L R R Q  600 48  CTAP&GCGCCC ACGAACCGTC TGAATCGGAG ACGATAAGAT CGGATGGTGG CACCTTCGAA E E T I D L A H E P S S R S G G T F E S  660 68  TTGTCCACTG GAGAGACAA CGAGGARTTA GAGTGCGCAG GCGTTGCCTT CTTCAGAAAG D N EEL E CA G VA F FR K L ST GE  720 88  ACGATCGAAA GCAACGCCAT CTTGTTGCCC CGATATCCCA GCGCCGATCT GTTGCTTTAC TIE L L P R Y P SAD L L SN AX L Y  780 108  GTTGTCCGAG GTAGGTTAAT ACATGATTGT GTATGGCACA TGATTGCCTA AAATTGTCAT intron 1 V V R  840 111  TATAATTGTG TATGCAG-TG AGGGCAGACT GGGAATTGTT TTCCCCGGAT GTCCGGAGAC  899  GE  G  R  I,  G  XV  F  PG  C  PET  126  TTTCAGAGAT CATTCCTCGT TTCAAGGGCG ATCAGGCAC AGATCAGAGG GACGACGGGA F RD H F SR H R SE G R RE S S Q G R  959 146  GGMGAGGAA GAGGAAGAAG AGGACTCAAG TCAGAGGTG AGGCGAGTGA GGAGAGGAGA R R V R R GD E E E HE E ED SS Q K V  1019 166  CGTAATAGCG ATATTTGCAG GAGCAGCCTA CTGGTCGTAC AACGAPGGCA ACGAGCCTCT Y ND G NE P L VIA IF A GA A Y W S  1079 186  CCAAATCGTA GGCATTGCCG ACACATCCAG CCGTCGAAAT CAGGGCCGCA GCAGGAGTTA SR G IA D T S S R RN S Y Q G R Q IV  1149 206  50  CCGCGTAAGA ATCCCGACCA ATTAACTAAT AATCATCTTC AGTTATATTA TAGATTTTTT R intron2  1199 207  CGTTTCTTTT ATAGTTGATT GATGGGGTAG AGATATATAC ATGTACAGCC CTTCTCTTTG P F S L  1259 211  GCTGGGCCAG GCTCATCATC TCGTCGTGAG GAGGGAGARG GA.AGCAAG AGGAATTGGG A C P C S S S R R E H E C C K G R C I C  1319 231  AGTAATATTT TTGCAGGTTT TAGCACTCGC ACTTTGGCTG AAACATTGGG GGTGGAGATT SN I FAG F S TR TL A ET L G yE I  1379 251  GAAACTGCAA GGAAGCTTCA AGAGAATCAG CAATCGCGAC TGTTTGCGAG CGTTGAPCGG ETA R IC L ENQ L FAR V ER Q Q SR  1439 271  GGCCAACGAC TGAGCTTACC CCGCCCTCGA TCTCGCTCTC GCTCTCCTTA CGAGACGGAG L P C L S P C R S R S R P Y S E R E Q R  1499 291  ACTGAGAGGG ATGATGTTGC TGGTGGATTG CAGGGATATT AT?CATCTGG AGATGACAAT T ER D D VA CCL Y SS C DEN Q C Y  1559 311  GGCCTTGAAG AGCTTCTGTG CCCACTGCGT CTAAAGCACA ATGCTGACAA TCCCGAGGAT P L R GV E EL V C VK H NA DN P ED  1619 331  CCCGATCTCT ACGTAAGAGA TGGGGGACGA TTGAATAGAC TCAACCGCTT CCTTCCT A D V Y V R D L N R V N R F C C R K L P  1679 351  GTACTCAACT ATTTCAGATT ACGAGCCGAG ACCCTTGTTC TCCACCCGGT AAGCAATAAC V L K Y L R L GA H R V V L HP  1739 367  TTTTTATTCG CTTCACTTAA TGTCAATTTT CAAGTCCAGT GAATGAATTA ATCTGGTTGC intron 3  1799  AGAGAGCATC GTGTGTTCCT TCGTGGAGGA TGAACGCGCA TGGCATAATG TACGTGACGA --R AS CV P SW R M NA H GIN Y VT  1859 386  GAGGGGAGGG GAGATTGAG GTGGTGGGAG ACGAGGCAG GAGCGTGTTT CATGCGCGTG R GE RI E G V V C D E CR S V F D CR  1919 406  TGAGAGAGCG TCAGTTCATC GTCATTCCCC A?TCTACGC ACTCATCAAA CAGGCAGGAG V RE C VIP V 1K Q F I Q F Y A Q AG  1979 426  GCCAGCGCTT TGAGTGGATA ACGTTCACAA CATCGCACAT GTAAGTATAA CATAATTAGC CE F E WI T F T T D I G S  2039 440  ATTGCACATG TCATGTACTG ATTGTTATAC TCATCATAAC TGGTATGCAT CTCAGTTCTT intron4 S  2099 441  TCCAGTCGTT TTTGGCGGGA AGGCATCAC TTTTGAAGGC ATGCCGGAG GAGTG?TGA F L A C V L R Q S K A N P H E V L Q S F  2159 461  51  GTGCCGCTTA CAGGATGGAC CGAACTGAG TCCGTCAGA TATGAGTAAC AGAGATGCG R T E V R Q I S A A Y R M D N S N R E C  2219 481  ACACCCTCAT TCTGCCTCCA TCATCCCT?G GACGTGACCA AGAACAACAG CACAACATCA D T I, I L P P S S L G R D Q E Q Q H N I  2279 501  CATCTCTTCTGCACCAAGTGGAgggcgt t tgaatgaatattatggaataaggcgt t t T S L L H Q V E gaatgaatattatcgagacagctctctgcttcacgcggtgtcctgtttgcgctgcat ggttcggcttagtagctagctacccaatattacaataaaacaatgataaggctgtaa -  509  tagatattataataaggatgttgctttctatgtgtctacaatttcgatggaactttc tccattatattcacatgcagctacgccctcagcgttttcgttttctccatatttcca aattccatcccaaagttataaaaca.tttgacgtgatttatatagcaaactcttttca  catggagcatgtcattaatgacctgggtttgtattaatattcttatcaaattaagaa aacactaccacatcggtcaaacattgtag  Nucleotide sequence of genomic DNA clones Fig. 4.4 S3.7 and E2.8 from Picea glauca uS legumin storage protein, and deduced amino acid sequence. The +1 site and the begining of the cDNA (=) are indicated. regulatory sequences are underlined. The Putative aminoacid sequence is printed below the nucleotide sequence. The nucleotide and amino acid sequences corresponding to the deletion are printed in italic characters. The coding region is printed in bold The aininoacid characters. Introns are indicated. sequence is explained in fig 4.7. On the 3’ non-coding and the polyadenylation region the first stop codon signals are underlined.  52  4.3. STRUCTURAL ORGANIZATION OF THE PICEA uS LEGUMIN GENE In order to align the cDNA to the gDNA,  4 gaps were  introduced on the cDNA sequence corresponding to introns and 1 gap introduced on the gDNA that corresponds to a putative deletion.  The  sequence  complete  coding  of  the  region of  E2.8 ilS  the  plasmid  contains  legumin gene  from  the  Picea  glauca.  The coding region is interrupted by 4 short introns  of  1104,  67,  contains  and  least  at  subfamily  74  A  75  nt  respectively.  additional  one  legumin  genes,  intron,  and  two  The when  more  gene  Picea  compared  introns  when  compared to subfamily B legumin genes from angiosperms. first  two  introns  are  located  in  the  region  encoding the acidic or a protein subunit, are  located on  subunit. introns All  the  Introns 1  four  to  of  3  3  gene  or  in  correspond  3 protein  position (figure  to  4.5).  flanked by canonical border sequences their  is  their  elevated AT content compared to the surrounding exons.  Dicot  4.6)  sequence.  introns  average  A remarkable  have  monocots.  direct  an  repeats  detected  in  (fig  no  and  subfamily A legumin genes  introns are and  2  the  The  and the last two  the basic  region encoding  1,  of  to  feature  average AT  were  of plant  content  of  introns  72%,  versus  56%  in  The content of A/T within the Picea introns is in  68.1%  (67.7,  71.1,  67.5  and  64.0%  for  each  intron  respectively).  53  2  1  tata  4  3  aataaaa  Picea  2  1  tata  3  aataaaa  ‘aa-a\aa-rrss4 2  tata I  3  aataaaa I  I  B  Fig 4.5 Comparison of and  angiosperm  numbered  1-4,  A  I  us  legumin genes from Picea glauca  subfamilies  and  exons  are  A  and  B.  shaded.  The The  introns  TATA  box  are and  polyadenylation signals (aataaaa) are indicated. The introns interrupt the coding regions at the same relative positions in each gene, but are variable in size.  54  Eukaryote consensus  GTA/QAG  -/G  GTAGGT GTCCAT GTCCAT  INTRON 1 67 nt 134 nt 237 nt  GTATGCAG TTTTATAG ATGAATAG  Radish Soybean A Soybean B Pea B  GTAAGA GTAATC GTGAGA GTGAGC GTAAGT  INTRON 2 104 nt 96 nt 282 nt 270 nt 75 nt  ATGTACAG TGCTGTAG CTTGGCAG CTTGGCAG TTTTTCAG  Picea  GTAAGC  Radish Soybean A Soybean B Pea B  GTAAGT GTACGT GTACGT GTACGT  INTRON 3 74 nt 265 nt 624 nt 395 nt 80 nt  GGTTGCAG TCTTTCAG TGGTGCAG CGATGCAG ATATGC  GTAAGT  INTRON 4 75 nt  CATCTAG  Picea Radish Soybean A  Soybean B PeaB Picea  Picea Radish Soybean A Soybean B PeaB  intron of uS legumin Comparison flanking Fig 4.6. sequences. The uS legumin gene introns are flanked by canonical border sequences. The size of each intron is indicated in nucleotides. Radish and Soybean A correspond to subfamily A legumin genes and Soybean B and Pea B to subfamily B genes.  55  The gDNA  gap  introduced when  aligning  the  cONA  to  the  showed a deletion that is present in the genomic DNA.  The 77 nucleotide sequence is located in the region encoding the  Cx  subunit.  sequence,  Among  are  4  the  highly  26  conserved  absent on the genomic DNA.  the  rest  amino  among  acids  legumin  of  this  genes  but  The deletion shifted the sequence  No other deletions or stop signals were found  out of frame. in  deduced  of  the  gene.  The  fact  that  this  is  the  only  deletion on the sequence and the fact that the positions of 3 of the four introns are conserved relative to angiosperms, that  suggests  deletion  A Hindlil  subcloning. c]DNA  the  within  the  may  be  restriction  sequence  that  an  artifact  site was  was  absent  of  located on  the  clone. To examine this possibility the other clone,  the  on  genomic the S3.7  plasmid DNA was tested for the presence of Hindill site fig 4.2). the  (see  The S3.7 contains a 2.3 Kb fragment that overlaps  E2.8,  Hindlil  the  however site  it  is  does  not  not  have  present  on  the the  Hindlil k—DNA  site.  The  either,  as  corroborated by enzymatic digestion and mapping of the k-DNA (fig  4.2).  cloning  These  artifact  propagation.  If  results that this  genome and this gene the  could is  a  have real  that  arisen  this  recent  with  the  deletion  in  cloning  The are  the  the  be  a  phage  original  is a pseudo gene this would have been  event based  cDNA.  could  during  first deletion accumulated in the gene,  very  from  suggested  on  the  high  possibilities difficult  to  of  and probably a  sequence an  explore  conservation  artifact because  arising it  would  56  require re-screening the library and obtaining  exactly the  same gene.  4.4. PICEA GLAUCA uS LEGUMIN AMINO ACID SEQUENCE Because of the deletion present on the third exon two different approaches can be taken to analyze the amino acid sequence deduce  the  using  the  amino  genomic  acid  DNA  sequence  includes the deletion or b)  nucleotide from  the  sequence  genomic  :  DNA  a) that  substitute the deleted region in  the genomic with the corresponding cDNA nucleotide sequence and  deduce  the  amino  acid sequence.  For  the purpose  of  this study the second approach will be used, because if this deletion  is  deletion,  which  intact and is  real,  it  means  would that  the  be  the  rest  first  of  the  accumulated gene  therefore suitable for comparison.  rest of the gene,  promoter,  coding region,  remains  Also,  the  intron positions  and intron / exon junctions are consistent when compared to angiosperms. Furthermore since there are no other gymnosperm SSP  gene  sequences,  this  presents  a  opportunity  good  to  study gene structure and to compare the gene and its product to genes and proteins  from angiosperms.  this  a  deletion  being  cloning  The possibility of  artifact  is  also  to  be  considered. If approach  “a”,  is  studied,  the deduced amino acid  sequence is truncated and the protein length. an  area  aminoacids  in  The 77 nucleotide deletion is in the third exon,  in  of  highly  conserved  is  244  aminoacids.  However  by  57  substituting sequence, protein  the deleted area with the  the product of the gene is a typical  comparable  to  other  amino acid sequence codes (Fig  4.7).  comparison 24  The  hydrophobic  Leal,  from  N-  codon  terminus,  the  ,  same  rice  may  site was  for a protein of  initiation  the  and  legumin proteins.  uS  legumin  The  deduced  509 amino acids  was  determined  legumin aminoacid sequences.  region  1993),  cleavage and  at  G1uB  and  ATG  to other  aminoacids  GluA  corresponding c]DNA  correspond  size  (Takaiwa  represent  as  et  the  estimated to  those  al,  to  first  a  very  reported  1991;  signal  The  Misra  peptide.  lie between  the  by  for and The  alanine-24  isoleucine-25 by comparison to the XI5H cDNA and to the  Douglas fir sequences. precursor  is  57.18  KD and the co-translationally processed  protein is 54.4 KD. legumin  subunit  The deduced molecular weight for the  The post-translational cleavage of the  precursors  into  ot  and  polypeptides  is  regulated by a protease that cleaves between asparangine and glycine residues  (Scott et al,  processing  precursors  the  1992).  of  uS  The cleavage site for legumin  proteins  in  angiosperms is highly conserved and the sequence surrounding this site is also well conserved. Recently it has been shown by  cDNA  sequence  that  the  predicted  cleavage  site  in  the  Douglas fir legumin-like gene is also between asparagine and glycine  (Leal  and  Misra,  1993).  The  legumin  gene  of  the  gymnosperm Gingko biloba, however, has an asparagine residue at  the  (Hager  N-  terminus  et  al,  of  1992).  the By  13  subunit  comparison  instead to  of  glycine  other  legumin  58  sequences,  the  spruce  amino  acid sequence  from 311  to  320  corresponds to the conserved sequence with the cleavage site between the asparagine and the glycine at positions 311 and 312,  respectively.  position 7 of the the  formation  f3  of  The  invariant  cysteine  residue  at  subunit has been shown to be involved in  a  disulfide  bridge  linking  the  I  and  subunits of uS legumins in angiosperms and in Gingko biloba (Hager et al, putative  In the spruce uS legumin protein the  1992).  cysteine  precursor protein.  is  located  at  position  318  of  the  The other cysteine that may be involved  in the formation of the bridge is at position 123 of the subunit,  x  determined by comparison to other legumin proteins.  As a result of the cleavage the  x subunit is a polypeptide  of 311 aminoacids with a molecular weight of 34.9 KD and the  I  subunit  is  198  amino  acids  in  length with a molecular  weight of 22.2 KID. The amino acid composition of the deduced protein is shown in table 4.1.  59  MQILLIASCFLFLSLSTVSPVTAI SQQRRGRGRRYDEQS S ScRBLRRLS  50  AHEPSESETIRSDGGTFELSTGEDNEELECAGVAFFRKTIESNAILLPRY  100  PSADLLLYVVRGEGRLGIVFPETFRDHS SFQGRSRHRSEGREEEEE  150  EEED S SQKVRRVRGD VIAl FAGAAYWSYND GNE PI QI VGIADTS SRNQ 1  200  GRSRSYRPFSIAGPGSSSRBEEGEGKGRGIGSNIFAGFSTRTI A 1 ETLGVE  250  IETAKLQENQQSRLFARVERGQRLSLPGPRSRSRSPYERETERDDVAGG  300  LQYYS SGGDENVEELIRVKHNDNPEDADVYVRDGGRLNRVNRFKL  350  PVLKYLRLGAERVVLHPRASCVPSWRAHGIMYVTRGEGRIEVVGDEGR  400  SVFDGRVREGQFIVI PQFYAVIKQAGGEGFEWI TFTTSDI SFQSFLAGRQ  450  SVLKMPEEVLSAAYRRTEVRQIMSNRECDTLILPPS SLGRDQEQQHN  500  ITSLIHQVE  509  Fig. 4.7 Spruce uS legumin deduced amino acid sequence. The protein sequence is in capital letters. The putative signal peptide the at N-terminus is underlined. The predicted cleavage site for the x and 3 subunits is shown C ‘1’). The two cysteins predicted to be involved in the formation of disulfide bridge are circled.  60  Arginine  11.7%  Aspartic acid 3.9%  Serine  9.7%  Phenylalanine  3 • 9%  Glutamic acid  9.3%  Proline  3.7%  Glyc me  9.0%  Asparangine  3.2%  Leuc me  8.1%  Tyros me  2.5  Valine  6 • 8%  Hi stidine  1.8%  Alanine  5.7%  Lysine  1.8%  Isoleucine  5.2%  Cysteine  1 • 6%  Glutainine  4.3%  Met ionone  1.6%  Threonine  3.9%  Tryptophan  0.5%  Table 4.1 Amino acid composition of the Picea uS legu.min protein.  61  4.4.1.  AMINO  ACID  COMPARISONS  REVEAL  PRESENCE  OF  compared  to  HIGHLY CONSERVED SEQUENCES  deduced  The  amino  acid  legumin amino acid sequences dicots,  and  to  two  to  EMBL  CDPROT18,  Intelligenetics,  protein  and PCLUSTAL programs (a and b)  was  from angiosperm,  gymnosperm  compared  sequence  sequences.  data  banks by  Inc.CA)  (fig 4.8).  The  monocots  and  sequence  was  (PCGENE  using  the  The results  release PCOMPARE  in table 4.3  are shown in terms of percentage of identity. The  results show 69.2 % identity between spruce and Douglas fir; 68.2 % between spruce and pine and 63.9 % between D.fir and pine.  The  percentage  dicots varies 39.8  %  within  from 28.8 different  same subfamilies. monocots  is  of  31.1  identity %  to 34.5  between %  subfamilies  the  spruce  and among dicots to  65.8  %  and from  within  the  Similarly the identity between spruce and %  to  34.5  %.  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The alignment was done on protein sequences using the CLUSTAL PCGENE. 8 Character to show that a position in the alignment is perfectly conserved: 1*1; Character to show that a position is well conserved: ‘‘.  65  PERCENTAGE OF IDENTITY AT THE AMINO ACID LEVEL a) D. Fir  Spruce  Spruce  D.  Pine  69.296  68.2%  Fir  63.9%  b)  Spruce  Soy 2  PeaA  Cot 2  Arabl2  Soy2  PeaA  Cot2  Arabl2  Ricel  29.7%  28.8%  33.7%  34.5%  34.5%  31.1%  65.8%  41.2%  39.8%  37.1%  37.5%  41.2%  41.3%  35.1%  36.1%  44.1%  40.5%  36.9%  39.6%  38.6%  Rice 1  Table 4.2 Percentage of identity at amino acid between a) Picea glauca and other gymnosperms; b) versus dicots and monocots.  Rice2  62.9%  level Picea  66  THE PICEA GLAUCA LEGUMIN PROMOTER REGION  4.5.  The indicate  that  region. using  restriction  The a  this  cap  site  at  embryo  scanning  for  the  1.4Kb  determined  that  was of  developmental  showed  sequence  contains  was  eukaryotic  program  of  by  the  primer  cDNA.  stages  were  of  the  S3.7  flanking extension  using  mRNA  elements  position  the  5’  constructed  promoter  the  of  27  from  three  assayed.  The  using  EUKPROM,  start  site  and  being the same as determined by primer extension.  TATA box, The  and  the beginning  different  PCGENE  clone  primer  27mer  nucleotides  map  start  site  upstream of  (÷1)  was  the ATG  (fig  determined 4.9  to  be  and 4.10)  97  nucleotides  and 35  nucleotides  downstream from the putative TATA box (fig 4.10).  4.5.1. PUTATIVE REGULATORY SEQUENCES Sequences gene has  responsible  for  the  regulation  expression have been described been  region  shown  (Shirsat  almost  et  investigate  the  sufficient  are  expression  that  al,  first to  equal 1989;  to  give that  Itoh,  the presence of  600  et  nt a  of  al,  known  region  conserved  were  sequenced.  motifs  were  of  the  level the  5’  of  complete  1993)  .  In  and  it  flanking regulated promoter order  to  that could be  450 nucleotides of the  Sequences  found  legumin  angiosperms  similar elements  regulating gene expression in spruce, promoter  in  of  in  the  homologous 5’  to  flanking  67  sequence and the results are shown on figure 4.10 and table 4.3. for  The cap site and TATA box were determined by scaning eukaryotic promoter elements using the  PCGENE program.  The putative TATA box is located at 35 bp from the cap site. that has been described as  One ACACA element, element  for seed specific position -208.  found at  expression on albumin genes,  was  Two  the  respectively,  the  region  upstream  et  (Depigny-This,  legumin  and are  of  al,  other  ABRE  (Marcotte et al,  elements 1989)  have been  shown  consensus  sequence  are located at -40  storage  seed  protein  the  to  RY-repeat  and rice  bind  to  were  first  described  factors.  transcriptional  included within  is  et al,  (Mundy J.  the  G-Box  sequence,  which contain ABRE elements at positions -132, another  recently  one  at  identified  thought  to  position.  -159 in  binds nuclear proteins position -216  the  An  sunflower  (Nunberg et al,  of the Picea promoter  be  at was  involved  in  binding  .  AGATGT  promoter 1994)  present within the -223  in  -  wheat  1990)  and  The ABRE sequence. three of  -137 and -109 element,  region  that  is present at  AGATGT elements are nuclear  enhancing expression of sunflower helianthinin. region is  genes  (ABA regulatory element)  Four G-Box elements are present on this  and  in  ABRE elements are present at -138 and  found and other two These  with  similar to those present  Close  1992).  position -110 one ABRE element  160.  boxes  or RY repeat,  consensus sequence CATGCAT, and -87,  an important  factors  and  An A/T rich  to -340 nucleotides.  A/T  rich regions have been implicated in the binding of nuclear  68  factors  (Meakin  described  Gatehouse,  1991;  to  the  high  similarity  in angiosperm SSP genes,  sequences are  et  al,  1993;  to  promoter  these various  elements conserved  likely to be involved in Picea SSP regulatory  functions which have yet expression  of  regulatory  elements  acting factors.  Itoh,  1994).  Nunberg et al, Due  and  a  specific and  to be characterized. gene depends a  specific  on  a  However,  the  combination of  complement  of  trans—  Table 4.3 shows the possible roles of Picea  elements compared to  elements in other ssp genes.  69  Determination  Fig.4.9. (+1)  by primer extension. and pre-enthryo  (lanes3-5) RNA  (lane  indicates right  of  of  1) the the  was  used  +1  site,  S3.7  DNA  the  n1ENA5  transcription  from spruce mature  (lane 2) as  and  sequence.  site  enthxyo  stages were assayed.  negative the  start  control.  sequence The  is  The  shown  TATA box  No  arrow at  sequence  the is  also shown.  71  uS LEGUMIN PROMOTER  (-87) (-40)  LEG BOX (RY) (-208) ACACA  (-35) TATL  ATG  aataaa  II Af\ (-223---340)  \  AGATGT (-216)  ABRE G-BOX (-109) (-132) (-137) (--159)  Fig. 4.10. Location of putative regulatory sequences on the promoter of Picea uS legumin gene. Isluinbers on the figure are relative to the cap site. Abbreviations are referred to in the text.  72  PUTATIVE REGULATORY SEQUENCES ON THE PICEA LEGUMIN PROMOTER CONSENSUS SEQUENCE  POSITION FUNCTION IN PICEA  TATA  -35  TATA-binding  BINDING FACTOR  REFERENCE  TFIID1,2  Chaiziberland, et al (1992)  basal transcription -40 LEG BOX RY repeat -87  CATGCAT ABRE/ G-BOX ACACGTG  -109 -132  :  ACACGTCG  ACACGACA  Tissue specificity  Lelievre, et al, (1992)  Enhancer of expression  Chamberland, et al (1992)  ABA  EmB-1  responsive  (1990)  element and  Mundy et al,  GcBT-1  Guiltinan, et al (1990)  Expression enhancer  ACACA  -208  Albumin seed specific expression  AGATGT  -216  Tissue specificity Legumin  -223 to -340  AfT  Seed nuclear proteins  expression  from  enhancer  soybean  Seed specific expression enhancer  LABF1 Nuclear factors of early embryo  Nunberg et al, (1994)  Meakin and Gatehouse, (1991) Itoh, et al (1993)  genesis Fig 4.3 Putative regulatory sequences in the Picea glauca  us  legumin promoter. Consensus sequence and positions refer  to the P. glauca promoter sequence. The function and binding  factor columns refer to other seed storage protein promoter sequences, from the literature.  73  Chapter 5  DISCUSSION  The coding region of the XI5H-1 Picea glauca uS legumin gene  characterized  homology with genomic have  DNA.  been  genomic  the  in cDNA  The  the  in  cDNA  present  clone  important  indicated  and  the  XI5H,  figure  in  4.4.  exhibits to  the  98.7%  isolate coding  the  this  region  Comparison  suggests  sequences  the coding region of the  used  features  (fig 4.4 and 4.5)  four introns  study  of  the  presence  of  and a short deletion within  Picea uS legumin gene.  Based on amino acid identity the existence of two legumin subfamilies subfamily  (A and B)  has  a  characteristic  position  of  the  two  of  number  number is two for B and three for A The  in angiosperms.  has been shown  introns:  (Boutler et al,  introns  subfamily  of  Each this  1987).  B  genes  correspond to the position of the second and third introns of subfamily A genes. The position of the introns is highly conserved  among  sequences  (Galau et al,  uS  legumin gene  legumin  genes  1991).  This  from Picea has  compared to angiosperm genes.  are  as  one  the  surrounding  study shows or  two  that the  extra  The position of  introns  introns  one  to three correspond to the position of one to three introns subfamily  A.  This  conservation  of  intron  gyrnnosperms.  Since  of  this  is  result  demonstrates  positions the  first  is report  that  the  extended  to  of  a genomic  74  clone  from  a  subfamily,  A  storage  or  based  however  speculated, type  seed  protein  gene  from  a  the possibility that this gene is a member of a  gymnosperm, different  legumin  B  the  genes  on  the  possibility  can  not  be  of  introns  is  containing  Evenmore,  to  (one  of  Picea  excluded.  intron positions  conservation of  number  the  suggests  three)  that these genes may have derived from a common ancestor. The possibility that the Picea uS to  different  a  percentage angiosperm  of  subfamily  homology  sequences.  is  at  The  legumin gene belongs  also  the  suggested  amino  percentage  acid  of  by  the  level  identity  with  between  subfamilies A and B in angiosperms of the same or different species  is  about  40%.  members of subfamily A, to 85%.  The  percentage  of  identity  among  of same or different species is 65%  The percentage of identity among dicots is higher  than it is between dicots and monocots. The analysis of the predicted amino acid sequences the  percentage  Picea  and  of  identity  monocots  percentage of  is  from this study shows  between  similar  Picea  and  dicots Since  (30-34%).  suggest  that  they  belong  to  three  or the  identity between Picea and subfamily A  not different than that between Picea and subfamily B, could  that  is this  different  subfamilies. Nevertheless the differences may be the result of  evolution.  gymnosperms This  The percentage  Picea,  Pinus  and  of  identity among  Pseudotsuga  is  the  around  three 70%.  high percentage suggests that they may belong to the  same subfamily. However no data is available at the genomic 75  DNA  level  for  Pinus  and  Pseudotsuga,  so  comparisons  regarding intron number can not be made.  Homologous regions among legumin amino acid sequences are dispersed  throughout  similarity  is  due  to  (Negoro et al 1985). confirmed  by  the  molecules,  divergence  suggesting  from  a  common  1988)  the  existence  Furthermore,  .  such  gymnosperms  of  structural  speculated  biloba  monocots gene  immunological  the  genes  dicots  and  sequences  (Templeman et  the presence of legumin proteins in Gingko  as  and  that  ancestor  homologous  and  conifer  supports the hypothesis of a common ancestor. of  the  The ancient origin of legumin genes is  expressed in the spores of some fern species al,  that  have  criteria  encoding evolved  On the basis it  legumin  from  a  species  has  been  proteins  common  in  ancestor  (Negoro et al 1985; Borroto and Dure 1987).  Recently molecular  evolution data  strongly suggest  that  the separation of monocot and dicot lineages took place in late Carboniferous  (300 million years ago).  time  from  of  million  conifers years  and  ago,  upper Devonian-Lower ago) (Martin that  the  et  al,  legumin  angiosperms the  earliest  Carboniferous  1993). gene  The  from  similar  to  angiosperm  genes.  introns  is  different,  the  is  age  plants  (360 of  though of  of  330  are  of  million years  this  glauca  Even  position  estimated  seed  results  Picea  The divergence  is  study  structurally  the  three  show  of  number the  of  four  introns is conserved among the gymnosperm Picea glauca and 76  angiosperms.  The results also show that at  the protein  level  shares  common regions  the amino acid  with a  30-34%  of  identity to angiosperm legumin proteins and 70% of identity with  other  gymnosperms.  that  shows  is  the organization of  between  similar  This  conifers  conservation of genes  first  the  report  the legumin gene  and  angiosperms.  and proteins  support  that  is highly  The  notable  the hypothesis  of a common ancestor. extent  The  of  conservation  uS  among  proteins  may  be  based on functional constraints to evolutionary divergence, the postranscriptional processing event including the  i.e.,  bond  disulfide signal  for  moving  to  formation,  moving the  the  through Golgi  proteolytic  the  the  ER,  and  apparatus  cleavage,  the  information  for  the  effective  accumulation of the proteins in protein bodies Dure,  (Borroto and  1987; Negoro et al 1985).  Besides genomic  clone  deletion region.  and  resides The  therefore  another  introns,  the in  the  deletion  the  cDNA  predicted  truncated protein.  is  third  moves  found  difference  the  amino  77  a  exon,  bp a  deletion. highly  sequence acid  between  out  product  the The  conserved of  would  frame, be  a  The deletion may be a cloning artifact.  However this possibility is difficult to explore because it would be necessary to re-screen the library and obtain the same  particular  gene.  The  probability  of  including  a  specific DNA sequence in a library with a known genome size 77  is described by the formula N=ln(1-p)/ln(1-f),  where p is  the probability of containing any particular DNA sequence, f  is  the  fraction and  fragment fragments  of  and,  For  recombinants  required  106.  to  the  Another  any  important  number  family members  each cloned  number  of  glauca  the  number  of  given  DNA  sequence  is  consideration  legumin genes belong to multigene families. gene  by  of  necessary  Picea  have  represented  the  to  therefore  clones.  x  genome  corresponds  N  recombinant  2.30  the  is  that  The number of  for Picea has not been characterized.  Nevertheless results  from this study show the presence of  at least two members of the legumin family (fig 4.1).  It is  important to mention that unsuccessful efforts were made to determine the number of the members of the However,  because  the  deletion  within the Picea sequence, repeats  or  palindromic  is  the  legumin family. only  one  found  and it is not bordered by direct  sequences,  the  possibility  being a cloning artifact has to be considered.  of  it  In animals,  bacteria and plant genomes it has been shown that there is a close correlation between direct repeats and occurring  deletions  upon  cloning  into E.coli,  naturally and  led  to  the authors to favor ‘slipped mispairing’ as a mechanism to explain  deletion  feature  that  artifact  is  formation  suggests that  the  that rest  (Heim this of  et  al,  deletion the  gene  1989). is has  Another  a no  cloning other  modifications and the promoter region appears to be intact. The amino acid identity,  if the deletion is substituted by 78  the  corresponding  sequence  within gymnosperms  from  cDNA,  the  is  very  high  and around 35% with angiosperms.  (70%)  The intron positions and the intron border sequences, insinuate  that  rules  for  the  However  the gene  is  eukaryotic  the possibility  functional,  this  they observe  flanking  intron/exon  of  since  being  the  sequences.  first  event accumulated in the Picea uS legumin gene, very  a  recent  even  Nevertheless, the  probably  pseudogene if  first  can  the  not  deletion  deletion  entirely is  in  deletion therefore  discounted.  real,  occurring  since  this  gene  the  would and  characteristics  still  can  be  for  used  is to  the rest  the  conserve  it  gene,  judge from the high sequence homology to the cDNA, of  also  original  comparisons.  The  results of this present work are important in the light of the  structural  organization  there is no data,  of  (C.  gene  other than that presented here,  genomic SSP DNA in gyrnnosperms. available  legumin  the  Newton,  since  regarding  Since the c]DNA sequence is  unpublished  results)  the  deleted  sequence was substituted by the cDNA corresponding sequence for the purpose of comparing to other legumin genes.  All  the amino acid comparisons were performed after translating the gDNA with the substituted sequence.  Other  important  features  found  in  the  legumin gene are found in the promoter region Table  4.3).  presence  of  The  results  putative  of  this  regulatory  glauca  Picea  present  (Fig 4.4 and  work  sequences  us  in  show  the  the  5’ 79  flanking  sequence  transcriptional presence  of  of  sequences  specific  legumin box,  ABRE  A/T rich regions,  as  sequences  presence  the  like  TATA  the  ACACA elements,  functional  have  The  and  similar  presence  in angiosperm SSP genes, role.  of  box,  highly  in  raise  basic  and  the  conserved  G-boxes  and  the possibility  regulatory of  Picea  sequences  various  are highly conserved and important  that  as  such  The  support the hypothesis that the Picea us  gymnosperms  angiosperms.  gene.  elements,  legumin gene may be that  the  for  to  sequences  transcription  strongly suggest a transcriptional  Some of these putative sequences have been determined  enhancers  factors Kahl,  in  or  variety  1991)  The  .  that  suggests  binding  the  of  elements  seed  for  specific  genes  presence  of  gene  functional.  is  transcriptional  these  (Weissing  conserved The  and  sequences  objectives  of  this thesis do not comprise the functional characterization of  promoter  the  interesting  to  region.  test  functional or not.  However  whether  the  it  would  promoter  and in a transgenic  directs  the  expression  of  the  genetic  transformation  of  plants  transfer of and  chimeric genes  Charest,  developed  for  microprojectile  very  sequence  is  The functionality of the promoter could  be examined by dissecting the gene promoter, reporter gene,  be  1991)  .  conifers  The at  system determine  gene.  into  Recent  have  made  if  it  advances  in  possible  conifer genomes  somatic  may  and  overcome  the  some  the  (Duchense  embryogenesis  B.C.Research  DNA-delivery  fusing it to a  system use of  of the 80  limitations  for  transgenic  tree  recovery  (Ellis  et  al,  1991)  The results of the present study showed that all the structural are  elements  similar  additional  to  of  the  Picea  angiosperms,  intron.  identity suggest that the subfamily  different  Another possibility two  introns  during  intron  angiosperm  that  fern  from  angiosperms) that  may  dicot  evolution  species  promoter  to  of  significant genetic  angiosperms  economically  exception  and  and  the  unique  to  A  lost gene  Picea  an acid  or  B.  one  or  is  more  The conservation of legumin seed plants  (gymnosperms  consequences  technology  (i.e.,  important  region  to  amino  subfamilies  tobacco,  species  also  exhibits  rule  out  the  all  gymnosperms,  it  is  regards developed  Arabidopsis)  such  presence  in  largely  as  conifers.  putative  elements associated with angiosperm SSPs. possible  the  of  and  strongly suggest an evolutionary relationship  have  application  legumin gene  angiosperm genes  similar to the ancestral gene. genes  the  us  Picea gene belongs to a legumin  than is  with  extra  The  glauca  of  in to The  regulatory  While it is not  regulatory  likely  the  that  sequences are common between gymnosperms and  domains  cis-acting angiosperms.  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