Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
THE EVOLUTION OF ALLELE DIVERSITY IN ANEUPLOID SPECIES OF CLARKIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0083157
Grant No.
(N/A)
Project No.
CA-D*-EVE-4066-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2001
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Project Director
Gottlieb, L. D.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
EVOLUTION AND ECOLOGY
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20124991080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1080 - Genetics;
Goals / Objectives
1) Sequence alleles encoding allozymes of PGIC having the same and different electrophoretic mobilities from numerous individuals and populations of four species of CLARKIA. It is expected that 20 or more alleles will be sequenced in each species. 2) Construct a tree of their genealogical relationships. 3) Determine the amount and type of sequence divergence within and between each of the species.
Project Methods
Standard protocols of molecular systematics and nucleotide sequence analyses will be used in this project as previously described (e.g., Gottlieb and Ford, 1996, 1997; Ford and Gottlieb, 1999). Large fragments of PgiC1, approximately 1000 bases long, will be obtained by PCR amplification of genomic DNA using available oligonucleotide primers designed to target various exons. Initially, a fragment between exons 10 and 14 will be amplified and both strands sequenced. This region is selected because it includes large introns that may differ in sequence among alleles, and will serve to distinguish PgiC1 from the pseudoPgiC2 should it be observed. Sequences will be determined by a Perkin-Elmer ABI Prism 377 Sequencer using standard vector primers. Large stocks of seeds of numerous populations of each of these species are already available from previous collecting efforts (Gottlieb and Janeway, 1995; Gottlieb and Ford, 1999).

Progress 10/01/01 to 09/30/06

Outputs
This project described the genealogical relationships of a large number of PGIC1 genes in a very closely related group of species of CLARKIA (Onagraceae), a genus that has long served as a model to understand evolutionary divergence and speciation in annual plants. Five diploid species distributed along the western foothills of the Sierra Nevada in California, and a very widespread allotetraploid species derived following hybridization between two of them, were examined. Previous studies had shown that the genomes of three of the diploid species had been restructured by chromosomal rearrangements as well as aneuploid reduction in chromosome number. Two of the species have seven pairs of chromosomes, one has six pairs, and two others have five pairs. Reduction in chromosome number is a step-wise process and one that provides evidence of phylogenetic direction; thus, one of the species with seven pairs is thought to have given rise to the one with six pairs which, in turn, gave rise to the two with five pairs, the latter arising independently. Since aneuploid reduction restricts the number of genes received from the progenitor at any locus to no more than two, and since the origin of these species appears to have been relatively recent, their gene lineages, in principle, could be connected to particular ancestral genes in the progenitor species. PGIC1 is a very well characterized nuclear gene that encodes the cytosolic isozyme of phosphoglucose isomerase which catalyzes an essential reaction preceding sucrose synthesis in the cytosol of plant cells. To date, a fragment of 1400 bases that includes portions of five exons and the introns between them has been sequenced from 39 PGIC1 genes. The genealogical network constructed from them permits the following conclusions: 1) Both species with seven pairs of chromosomes are monophyletic; i.e., all alleles examined within each form a single clade; 2) The species with six pairs was derived from the common ancestor of the two species with seven pairs and not from one of them as previously thought; 3) The genes in the six-paired species were extremely diverse (the most distinct differ by about 7.6 percent of their nucleotides) and were placed into four subclades; 4) Genes from the two species with five chromosome pairs clustered separately among the four subclades indicating they were derived from different ancestral genes consistent with the hypothesis that they arose independently, and that the six-paired species is polyphyletic; 5) The genes from one of the five-paired species formed two subclades and the genes from the other formed a single subclade, both results consistent with the genetic bottleneck consequent to their aneuploid origins; 6) The allotetraploid species contained two PGIC1 genes, one derived from one of the seven-paired species and the other from one of the five-paired species as predicted from the previous cytogenetical study.

Impacts
Following the origin of a species, ancestral gene lineages are gradually lost and new lineages arise. The result of such gains and losses is that eventually all genes of a new species will be more closely related to each other than to any in the sister or progenitor species. The sorting is a straightforward consequence of various microevolutionary processes (selection, mutation, drift, gene migration, population size fluctuation) and is well understood theoretically, but there are very few descriptions of gene lineages in wild plants. Such information is relevant to understanding the origin of genetic diversity and may be particularly applicable to the conservation and management of the small populations characteristic of many endangered species. The genealogical analysis of PGIC1 has provided one of the clearest pictures available of the build up of genetic diversity during the evolution of plant species.

Publications

  • No publications reported this period


Progress 01/01/03 to 12/31/03

Outputs
My laboratory continues to study molecular evolution of PGIC genes in CLARKIA, a genus of annual wildflower species nearly all native to California. PGIC genes encode the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9), an enzyme that catalyzes an essential reaction during glycolysis and gluconeogenesis. The present project uses genealogical analysis to examine the relationships and diversity of PGIC genes in a group of closely related species of the genus. Two of the species have seven pairs of chromosomes, one has six pairs, and two others have five pairs. Cytogenetic studies showed that one of the species with seven pairs gave rise to the one with six pairs which, in turn, gave rise to the two with five pairs by processes of chromosome rearrangement and aneuploid reduction. In principle, species origin by aneuploidy restricts the number of genes received from the progenitor at any locus to no more than two. When such a bottleneck occurred sufficiently recently that lineage sorting along species lines has not taken place, it may be possible to identify by sequence analysis the hypothetical genes of the progenitor that are the source of genes in the derived species. For this project, we are sequencing a fragment of PGIC that includes 1400 base pairs containing both exons and introns. During 2003, 13 genes were studied from the five species. In addition, we examined six PGICs from a widely distributed allotetraploid species that arose following hybridization between one of the species with seven pairs of chromosomes and one with five. The 19 genes supplement 17 that were examined during 2002. A network of genealogical relationships among the 36 genes showed that those from the two species with seven chromosome pairs constitute a pair of clades that are distinct from the genes from species with six or five chromosome pairs, as expected. Alleles of one of the two PGICs from the tetraploid cluster with genes from one of the species with seven pairs and alleles of the other PGIC cluster with genes from a species with five pairs, also as expected. Overall, the analysis shows that the genes from the three derived species, those with six or five pairs, are interspersed in a series of subclades and have not yet assorted along species lines, consistent with the presumed recent origins of these species. When our gene sampling is completed, the analysis is expected to provide an unusually clear picture of how a family of genes at a particular locus is built up during the evolutionary divergence of species.

Impacts
These studies contribute to basic knowledge about the genetic basis of species diversity and the origins of that diversity. The results may be applicable to the conservation and management of the small populations characteristic of these and many other endangered species.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
My laboratory continues to study molecular evolution of PGIC genes in CLARKIA, a genus of annual wildflower species nearly all native to California. PGIC genes encode the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9), an enzyme that catalyzes an essential reaction during glycolysis and gluconeogenesis. Our present project uses genealogical analysis to examine the relationships and diversity of PGIC genes in a group of closely related diploid species of CLARKIA, distributed along the western foothills of the Sierra Nevada of California. Previous cytogenetic studies showed that the genomes of three of the species have been restructured by chromosomal rearrangements as well as aneuploid reduction in chromosome number. The reduction in chromosome number correlates with geographical distribution. Thus, C. BOREALIS on the north has seven pairs of chromosomes, the ancestral number in the genus, and is thought to have given rise to C. MOSQUINII, with six pairs, now found in the central region. C. MOSQUINII is thought to be the progenitor of C. AUSTRALIS and C. VIRGATA, both with five pairs of chromosomes, and both found to the south. Origin by aneuploidy restricts the number of alleles received from the parent at any locus to no more than two. Such a bottleneck makes it possible to determine which gene lineages in the parent gave rise to genes in the derived species, thereby, providing phylogenetic order to nucleotide substitutions and information about how a family of genes is built up following the origin of a new species. The PGIC gene is complex with 23 exons and 22 introns in the coding region, extending over about 6000 base pairs. For this project, we are sequencing a fragment of 1400 base pairs between exons 10 and 14; to date, we have analyzed 17 genes. The genes were initially selected on the basis of the electrophoretic mobilities of their enzyme products, but both similarity or difference in mobility proved to be a very poor guide to sequence divergence and genealogical relationship. A tree of genealogical relationships showed that the PGIC genes in the three aneuploid species have not yet assorted along species lines, consistent with their recent origins. Thus, although genes from C. MOSQUINII, C. AUSTRALIS and C. VIRGATA constitute a clade distinct from that of C. BOREALIS and C. MILDREDIAE, a related species with seven chromosome pairs also found in the north, those of the two species with five chromosome pairs were nested among those from C. MOSQUINII. Additional sampling is underway to provide better understanding of the genealogical relationships between the gene lineages now in separate species and to assess the relative divergence among them.

Impacts
These studies contribute to basic knowledge about the genetic basis of species diversity and the origins of that diversity. The results may be applicable to the conservation and management of the small populations characteristic of many endangered species, and may also be useful to the design of programs to identify wild relatives and source populations of various crops.

Publications

  • Ford, V.S. and L.D. Gottlieb. 2002. Single mutations silence PGIC genes in two very recent allotetraploid species of CLARKIA. Evolution 56:699-707.
  • Gottlieb, L.D. and V.S. Ford. 2002. The 5 foot leader of plant PGIC has an intron: the leader shows both the loss and the maintenance of constraints compared with introns and exons in the coding region. Molecular Biology and Evolution 19:1613-1623.


Progress 01/01/01 to 12/31/01

Outputs
My laboratory continues to study molecular evolution of PGIC genes in CLARKIA, a genus of annual wildflower species nearly all native to California. PGIC genes encode the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9), an enzyme that catalyzes an essential reaction during glycolysis and gluconeogenesis. We are currently studying PGIC genes in allotetraploid species of CLARKIA. These species combine the genomes of pairs of related diploid species so that many of their genes can be expected to have similar sequences and similar functions. Our interest is to assess whether the possession of similar genes bestowed on polyploids as a consequence of their mode of origin leads to increased rates of gene silencing. For this purpose, we examined PGIC genes in the allotetraploid C. SIMILIS and its two diploid parents. The parents each have two expressed PGIC genes so C. SIMILIS was expected to have four. Complete sequences of the genes from both parents and from C. SIMILIS were obtained from a combination of PCR-amplified genomic DNA and cDNA from RT-PCR and 3' RACE experiments. Four PGIC genes were found as expected, but one of them was silenced by a single mutation that created a stop codon in an exon. An mRNA transcript is still synthesized, but any translation product would be severely truncated and without function. The silencing mutation probably evolved recently since the gene is not much different in sequence from its orthologue in the diploid parent and its base substitution rate is not accelerated relative to that of its orthologue or to other active PGIC genes in the same genome. We previously reported that one of the four PGIC genes in the related allotetraploid C. DELICATA was also silenced by a single mutation, in this case by a short deletion of an intron splice junction resulting in the synthesis of transcripts that lack an entire exon and, if translated, a defective protein. The discovery of such mutations would seem to support the hypthesis that gene silencing occurs in polyploids because they have multiple similar genes. However, the PGICs silenced in these two tetraploids were also silenced in various diploid species of CLARKIA and in a third tetraploid species of the genus the homologous PGIC remains active. Thus, the silencing in C. SIMILIS and C. DELICATA may have more to do with the properties of the particular PGIC gene than with tetraploidy. Genetic similarity need not imply genetic redundancy and many functions and interactions of the individual PGIC genes remain unknown. Further studies are required before concluding that genes in tetraploids are silenced because other similar genes are also present.

Impacts
Information about the presence and expression of specific genes in polyploid plants and comparison to the same genes in their diploid parents is relevant to understanding how polyploidy evolves, and may be useful to programs attempting to introduce and/or manipulate genes in polyploid crops.

Publications

  • No publications reported this period


Progress 01/01/00 to 12/31/00

Outputs
We study molecular evolution of PGIC genes in CLARKIA, a genus of 42 annual species nearly all native to California. PGIC encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9), an enzyme that catalyzes an essential reaction step in glycolysis. PGIC was duplicated in the ancestral stock of CLARKIA giving rise to paralogous genes PGIC1 and PGIC2. CLARKIA DELICATA, native to a limited region of southern California, is an allotetraploid species that combines the genomes of two diploid clarkias. As a result, the species has two homoeologous subgenomes that contain genes with similar functions. We wish to learn whether such genes are redundant and, consequently, subject to silencing, a problem of great relevance to understanding the basis of the evolutionary success of polyploidy in plants. The diploid parents of C. DELICATA each have an active PGIC1 and an active PGIC2 gene so that their tetraploid derivative has four PGIC genes. The sequences of the four genes were obtained from a combination of PCR-amplified genomic fragments and cDNA fragments from RT-PCR and 3'RACE that provided the complete coding regions and the 5'- and 3'-nontranslated regions of all the genes. Three of the four genes are completely normal and are presumably translated into active proteins. However, the fourth gene has a short deletion at the end of one of its introns that prevents normal mRNA processing with the result that the mature transcript lacks a complete exon. The absence of the exon removes 69 bases encoding 23 amino acids so that a functional enzyme cannot be produced. The defective gene is found in two populations, but a normal, functional allele that does not have the deletion and is otherwise identical to the defective one was found in a third population. This polymorphism of a defective and a normal allele in an allotetraploid plant species illustrates problems of recognizing when gene silencing has occurred and is relevant to defining genetic redundancy. Our study is the first to document the molecular basis of a silenced gene in a tetraploid plant.

Impacts
Information about the presence and expression of specific genes in polyploid plants and comparison to the same genes in their diploid parents is relevant to understanding how polyploidy evolves, and may be useful to programs attempting to introduce and/or manipulate genes in polyploid crops.

Publications

  • No publications reported this period


Progress 01/01/99 to 12/31/99

Outputs
We continue to study molecular evolution of PGIC genes in CLARKIA, a genus of 42 annual species nearly all native to California. PGIC genes encode the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9). During the previous year, we carried out studies of the genes in the allotetraploid species C. DELICATA and C. SIMILIS and their respective diploid parents. Although the parental species each express both PGIC1 and PGIC2 gene, a pair of paralogous duplicate loci, previous electrophoretic analysis had suggested that the tetraploid species have two PGICs rather than the four expected. However, analysis of genomic DNA sequences has now revealed that both tetraploids have two PGIC1s and tow PGIC2s, and analysis of cDNA clones showed that all four genes are transcribed in both. But, at least one of the genes from C. DELICATA encodes a faulty transcript that could not be translated into a functional product. In the portion sequenced to date (600 to 2100 nt per comparison), about half of the 56 substitutions occurred in the diploid parents and about half occurred in the tetraploid derivative. The absence of differences in the rate or type of substitutions between the ploidy levels is some of the first evidence that molecular evolution is probably similar in diploid and tetraploid plants, and thereby, addresses one of the central questions of plant evolution.

Impacts
Information about the presence, expression and interactions of specific genes in polyploid plants and comparison to the same genes in their diploid parents is relevant to understanding how polyploidy evolves. Such information may be useful to alter metabolism or development in polyploid crops.

Publications

  • Ford, V.S. and Gottlieb, L.D. 1999. Molecular characterization of PGIC in a tetraploid plant and its diploid relatives. Evolution 53:1060-1067.


Progress 01/01/98 to 12/01/98

Outputs
We continue to study molecular evolution of PGIC genes in CLARKIA, a genus of 42 annual species nearly all native to California. PGIC genes encode the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9); they were duplicated in the basal stock of the genus and about half of the diploid species have active PGIC1 and PGIC2 genes, whereas in other diploids only PGIC1 is active. During the previous year, we completed analysis of the genes in the allotetraploid C. GRACILIS and its diploid relatives. The tetraploid species was shown to have three loci: two PGIC1s and one PGIC2. All three loci are active and expressed. They are unremarkable in structure and sequence and appear to be evolving at similar rates to their orthologues in diploid species of the genus. The discovery of an expressed PGIC2 was unexpected because all related diploid species only have active PGIC1s. Thus, the diploid progenitor that provided PGIC2 is now extinct. CLARKIA GRACILIS is the first example of a tetraploid plant that retains an active locus no longer expressed in its diploid relatives. We are now examining PGIC genes in other tetraploids and their related diploid species of CLARKIA.

Impacts
(N/A)

Publications

  • FORD, V.S. and GOTTLIEB, L.D. Molecular characterization of PGIC in a tetraploid plant and its diploid relatives. Evolution. In press.


Progress 01/01/97 to 12/01/97

Outputs
We continue to study the molecular evolution of the duplicated PGIC genes in CLARKIA, a genus of 42 species nearly all native to California. CLARKIA includes four allotetraploid species whose diploid parents have been rigorously identified by previous morphological and cytogenetical studies. These species express two PGIC genes, yet their diploid parents have three or four active PGICs, implying that one or two of the genes were silenced or lost after their origin. Our present project will determine which genes are expressed in each tetraploid and if the inactive genes remain in the genome or have been lost. Expressed genes in experimentally synthesized triploid and tetraploid hybrids will be identified and compared with those in native species. Our studies are an attempt to understand at the molecular level the fate of particular genes encoding enzymes in the formation and establishment of allotetraploid plant species.

Impacts
(N/A)

Publications

  • GOTTLIEB, L.D. and FORD, V.S. 1997. A recently silenced duplicate PGIC locus in CLARKIA. Molecular Biology and Evolution 14:125-132.
  • GOTTLIEB, L.D. and JANEWAY, L.P. 1997. A new subspecies of CLARKIA MILDREDIAE (Onagraceae). Madrono. In Press.


Progress 01/01/96 to 12/30/96

Outputs
We continue to study the molecular evolution of the duplicated PGIC genes in CLARKIA, a genus of 42 species nearly all native to California. Two duplicated PGIC genes are expressed in about half of the ediploid species, but other diploid species express only one. Our recent phylogenetic analysis revealed that the PGIC duplication occurred prior to the diversification of all extant clarkias, which means that the absence of one PGIC in the latter species resulted in silencing. The recent discovery of an inactive, unprocessed pseudoPGIC confirmed this formal prediction. The pseudoPGIC includes more than 5000 neucleotides corresponding to 18 of the 23 exons and intervening introns of active PGIC, genes. It appears to be the first documented example of the inactivation of a specific duplicated locus that is normally expressed in related plant species. CLARKIA also includes a number of allotetraploid species whose diploid progenitors were identified by previous workers. Since the tetraploids do not express all of the PGIC genes that are active in their respective parents, we have initiated studies to find out which genes are active and which were lost or silenced as part of a larger study of genome interaction in tetraploid plants.

Impacts
(N/A)

Publications

  • GOTTLIEB, L.D. and FORD, V.S. 1996. Phylogenetic relationships among the sections of CLARKIA (Onagraceae) inferred from the nucleotide sequences of PGIC.Systematic Botany 21:45-62.
  • GOTTLIEB, L.D. and FORD, V.S. 1996. A recently silenced, duplicate PGIC locus inCLARKIA. Molecular Biology and Evolution. IN PRESS.


Progress 01/01/95 to 12/30/95

Outputs
Research continued on the molecular evolution of the duplicated PGIC genes in CLARKIA, a genus of 42 wildflower species, nearly all native to California. During the last year, additional nucleotide sequences were obtained, including a complete PGIC from an appropriate outgroup, permitting us to carry out a phylogenetic analysis of genes representing all the diploid sections of the genus. Parsimony analysis placed the sequences into two groups, PGIC1 and PGIC2, confirming that a single event ancestral to all extant clarklas accounted for all duplicate genes. The gene tree was very strongly supported by bootstrapping and the modified Templeton's tests. Fitch-Margoliash and neighbor-joining methods gave the same tree. The data for the two genes were completely concordant and establish the relationships among the sections. Both genes were expressed in species with two PGIC isozymes, but species with a single isozyme always have an expressed PGIC1 gene and a silenced pseudoPGIC2 gene. At least four independent silencings of PGIC2 occurred. Study of an unprocessed pseudoPGIC2 cloned from a genomic library of C. MILDREDIAE showed numerous flaws resulting from insertions and deletions, and loss or modification of intron-exon splice junctions, but no increase in nucleotide substitutions compared to expressed PGIC2 genes, suggesting its silencing was relatively recent.

Impacts
(N/A)

Publications


    Progress 01/01/94 to 12/30/94

    Outputs
    Research continued on the molecular evolution of the duplicated PGIC genes in CLARKIA, a genus of wildflowers mostly native to California. PGIC encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9), which catalyzes the reversible isomerization of fructose-6-phosphate and glucose-6-phosphate, an essential step in the conversion of triose phosphates to sucrose in the cytosol. During the last year, we obtained seven complete sequences and three partial ones. As a result, we now know that, following a single ancestral duplication, PGIC genes diverged to form two paralogous lineages designated PGIC1 and PGIC2. When a CLARKIA species expresses two PGIC isozymes, they are encoded by a PGIC1 gene and a PGIC2 gene. When a CLARKIA has a single PGIC isozyme, i.e., appears not to have the duplication, the single isozyme is encoded by a normal PGIC1 gene, but the genome also contains a duplicate PGIC2 gene that has been silenced by mutation and is no longer capable of expression. Two such pseudoPGIC2 genes were studied, revealing many exons with deletions and frame-shift mutations, many absent or partially deleted intron-exon splice junctions, and numerous insertions/deletions in introns. The sequence analysis also revealed that even when the PGIC2 gene is functional, it generally has a partial or completely deleted TATA box which, assuming reduced transcription rates, could account for the reduced activity of the PGIC3 isozyme relative to that of the PGIC1 isozyme.

    Impacts
    (N/A)

    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      Research continued on the molecular evolution of duplicated PGIC genes in CLARKIA, a genus of annual wildflowers native to California. PGIC encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9), an essential enzyme of glycolysis and gluconeogenesis. We examined duplicate PGIC genes from C. XANTIANA and the single PGIC from C. MILDREDIAE, species representing different sections of CLARKIA. The three genes had identical exon-intron structures to the previously studied C. LEWISII PGIC genes and were highly similar in sequence. Nevertheless, DNA distance and parsimony analyses revealed that the duplicate PGIC genes have diverged to form two lineages, C1 and C2, such that the C1 gene in C. XANTIANA is more closely related to the C1 gene in C. LEWISII than the C2 gene in its own genome and likewise for C2. Thus a single duplication accounts for the presence of two PGIC isozymes in the two species. The C. MILDREDIAE PGIC clusters with the C1 genes suggesting its genome may also have a duplicated C2 that was silenced and no longer encodes a protein. Recently a pseudoPGIC was identified in C. MILDREDIAE and it is currently being studied. By inference, the three species are monophyletic.

      Impacts
      (N/A)

      Publications


        Progress 01/01/92 to 12/30/92

        Outputs
        My laboratory studies molecular evolution of PGIC which encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9). This enzyme catalyzes the reversible isomerization of fructose-6-phosphate and glucose-6-phosphate, an essential reaction in glycolysis and carbohydrate metabolism in plant cells. We are examining PGIC in the wildflower CLARKIA, a genus of annual plants native to California because 12 diploid species of the genus have duplicated PGIC genes but another 12 have only a single copy, thus providing an opportunity to compare duplicate genes and their products with nonduplicate homologues in species with relatively similar genic backgrounds. Previously we cloned and sequenced both duplicate PGICs from C. LEWISII. They proved to have a complex structure of 22 introns and 23 exons. To determine if their structure was unusual, we cloned and sequenced completely the PGIC from the unrelated plant ARABIDOPSIS THALIANA. Its PGIC also proved to be highly structured with 21 introns. Their positions corresponded precisely to intron positions in the CLARKIA gene, but it had one more. The A. THALIANA protein is nine amino acids shorter than the CLARKIA ONE (560 VS. 569) and is 83% identical in its sequence.

        Impacts
        (N/A)

        Publications


          Progress 10/01/87 to 09/30/91

          Outputs
          The enzyme phosphoglucose isomerase (PGI; E.C. 5.3.1.9) catalyzes the reversibleisomerization of fructose-6-phosphate and glucose-6-phosphate, and is essential in glycolysis and carbohydrate metabolsim. In plant cells, this reaction is carried out by different isozymes in the cytosol and in the plastids, both isozymes encoded by independent nuclear genes. We have been studying the cytosolic PGI in CLARKIA, a genus of California wildflowers, in which the coding gene has been duplicated. We wish to understand how the PGI duplication originated, whether the rates and patterns of PGI nucleotide divergence differ in species with the duplication versus those with one gene, and the mechanism responsible for the similarity of PGI activity in CLARKIAS with one or two PGI genes. Our most significant results are that we have cloned and sequenced both duplicate PGI genes from C. LEWIS II and, since PGI has not been examined in other plants, a cDNA encoding PGI from ARABIDOPSIS THALIANA. In CLARKIA, the PGI gene has 23 exons and 22 introns, the highest number yet reported in plants, and encodes a protein of 569 amino acids. The protein is about 44-46% identical to the inferred protein sequences of pig, E. COLI and S. CEREVISIAE, and about 2, identical to the A. THALIANA PGI. Ours is the first report of a plant cytosolic PGI gene sequence. This research is basic to understanding the mechanisms of gene evolution and may prove useful to the eventual manipulation of carbohydrate metabolism in crops.

          Impacts
          (N/A)

          Publications


            Progress 01/01/90 to 12/30/90

            Outputs
            Two isozymes of PGI, an essential enzyme of intermediary metabolism, are generally present in plant cells, one in the cytosol and the other in plastids, both encoded by nuclear genes. In the wildflower CLARKIA, native to California, however, half the diploid species have duplicated cytosolic PGI isozymes. We are studying the PGI duplication to determine how it originated, whether the duplicate genes are regulated differently, and whether there are different patterns of genetic variation in duplicate versus non-duplicated genes. During this grant period, we cloned and sequenced two allelic PGI genes encoding one of the cytosolic isozymes of C. LEWISII, a species with the duplication. The genes show extensive intron-exon structure, with 19 exons so far identified encoding 88% of the amino acids (483/550) in the protein. The exact intron-exon borders were determined by sequencing PCR-amplified cDNA copies of poly(A) RNA isolated from descendents of the same plant used to make the genomic library. The two genes were shown to be allelic by matching cloned RFLPs from each of them to RFLPs in genomic DNAs prepared from segegating PGI genotypes. The genes are nearly identical in sequence with about 99% nucleotide identity in both introns and exons.

            Impacts
            (N/A)

            Publications


              Progress 01/01/89 to 12/30/89

              Outputs
              Molecular studies of glycolytic phosphoglucose isomerase isozymes (PGI; EC 5.3.1.9) in Clarkia (Onagraceae), a genus of plants native to California, were continued. We isolated and sequenced nuclear genes encoding the cytosolic PGI isozyme from C. xantiana, C. mildrediae and C. lewisii. Similar to a gene from C. unguiculata that we previously characterized, the PGI genes from the former two species lacked introns and could be expressed in E. coli as genomic clones. However, the gene from C. lewisii proved to have extensive structure, with at least 15 exons and introns. We now believe it likely that the intronless genes are processed genes that originated when cDNA copy of a mRNA transcript of a structured gene like that a C. lewisii was integrated into the genome in a position and orientation that permitted it to be expressed. We also identified a transposon-like structure within an intron of the C. lewisii gene and a number of direct repeat sequences in noncoding flanking regions of this and other Clarkia PGI genes, evidence that suggests the possibility that transposons could have facilitated movement of the postulated processed genes.

              Impacts
              (N/A)

              Publications


                Progress 01/01/88 to 12/30/88

                Outputs
                Molecular genetic studies of glycolytic phosphoglucose isomerase isozymes (EC 5.3.1.9) in Clarkia (Onagraceae), a genus of plants native to California, were continued. During the past year we sequenced, for the first time in plants, a nuclear gene encoding the chloroplast isozyme of PGI and one encoding the cytosolic isozyme. The former isozyme governs a reaction preceding the synthesis of starch and the latter one preceding the synthesis of sucrose. The identities of the genes were validated by comparison of their predicted amino acid sequences to recently published sequences for mouse and pig PGIs. The two Clarkia PGI genes are about 60% similar over their entire coding regions and each is about 65% similar to pig PGI. Neither Clarkia gene has introns and both can be expressed in E. coli. The PGI proteins synthesized in E. coli form dimers, are catalytically active and their electrophoretic mobilities are similar to those of appropriate Clarkia PGIs. Since PGI from a prokaryote has not yet been sequenced, and since to study the evolution of this gene a prokaryotic "reference point" was required, we also sequenced the PGI gene from E. coli. The E. coli PGI gene is 88% similar to the sequence of the plant chloroplast PGI and about 60% similar to that of the cytosolic PGI, a result consistent with the model that the chloroplast PGI gene was derived from a prokaryotic source.

                Impacts
                (N/A)

                Publications


                  Progress 01/01/87 to 12/30/87

                  Outputs
                  Genetic, biochemical and molecular studies of phosphoglucose isomerase isozymes (EC 5.3.1.9) in Clarkia (Onagraceae), a genus of plants native to California, were continued. About 12 species in this genus have a duplication of the nuclear gene encoding the cytosolic isozyme of PGI and for this reason previous studies have emphasized genetics and structural biochemical analyses. During the past year, we have constructed several genomic libraries from different Clarkia species and using a yeast PGI probe have cloned genes encoding PGI from four of them. Two genes have been completely sequenced. Their identities were determined by comparison of their predicted amino acid sequences to a previously published partial amino acid sequence of pig PGI. The two Clarkia genes are 59% homologous over their entire coding regions and each is about 65% homologous to pig PGI. Neither Clarkia gene has introns, and both can be expressed at high levels in E. coli. One of the genes encodes the cytosolic isozyme and the other the plastid isozyme. The sequences are being analyzed to learn how the duplication arose, how the duplicated genes are regulated, and to evaluate the usefulness of the gene sequences for inferring species phylogeny.

                  Impacts
                  (N/A)

                  Publications


                    Progress 01/01/86 to 12/30/86

                    Outputs
                    Genetic and biochemical studies of phosphoglucose isomerase isozymes (PGI; EC 5.3.1.9) in Clarkia, a genus of plants native to California, were continued. Previously, I reported the successful induction of mutations by ethyl methane sulfonate (EMS) treatment in each of the duplicated genes encoding the cytosolic PGI isozymes. A mutant of the nuclear gene encoding the plastic PGI has also been recovered. It was backcrossed to wildtype for five generations prior to characterization. Immunological analysis of homozygous derivatives revealed that the mutant accumulates only 50% of wildtype plastid PGI activity, and that the reduction in activity resulted in a 40% reduction in starch accumulation. The mutant plants exhibited a high level of sucrose in the leaves relative to wildtype presumably because the photosynthate normally directed to starch biosynthesis was exported from the chloroplasts. The plastid PGI mutant is one of the very few genetically characterized mutants that affect starch level in higher plants and, as such, its physiological consequences are under intensive study. Since studies of the evolution of gene loci require precise understanding of species phylogeny, restriction enzyme analyses of chloroplast DNA in Clarkia were also undertaken. The analysis in sect. Peripetasma, the largest section in the genus, provided an unambiguous and detailed genealogical history that will facilitate selection of PGI genes for our recently initiated nucleotide sequence studies.

                    Impacts
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                    Publications


                      Progress 01/01/85 to 12/30/85

                      Outputs
                      Genetic and biochemical studies of the duplicated cytosolic isozymes of phosphoglucose isomerase (PGI; EC 5.3.1.9) in Clarkia, a genus of plants native to California, were continued. We previously reported immunological inhibition and titration studies that revealed that species with the duplication have the same level of PGI activity and protein as those without the duplication, and suggested that some form of metabolic or genic regulation had evolved that compensates for it. In order to distinguish the two forms of potential regulation, we induced, by ethyl methane sulfonate treatment, mutations in each duplicated gene which caused complete loss of the activity of its enzyme product. The mutants were backcrossed to wildtype for five generations, thereby placing them on a 98.4% wildtype background prior to characterization. Homozygous mutants at one of the duplicated genes had 64% of wildtype activity and those at the other duplicated gene had only 36%. An F(2) progeny synthesized between the mutants revealed that the two loci had additive effects. Thus the double homozygous mutant was lethal, and one segregating class had only 14% of wildtype activity. Since the mutations sharply reduced PGI activity, the results showed that the activity is not directly regulated by metabolic factors. The near identify of PGI levels in species with and without the duplication probably evolved by changes in factors that regulate transcription or translation.

                      Impacts
                      (N/A)

                      Publications


                        Progress 01/01/84 to 12/30/84

                        Outputs
                        Genetic and biochemical studies of glycolytic enzymes in higher plants have beencontinued. The studies of triose phosphate isomerase (EC 5.3.1.1; TPI) have now been augmented by our recent report of the complete nucleotide sequence of the E. coli TPI gene. The gene encodes a polypeptide of 255 amino aicds and is 55% homologous to the previously sequenced yeast TPI gene. The predicted amino acid sequence of the E. coli TPI is approximately 46% homologous, on the average, to eukaryotic TPIs. The sequence information is consistent with the previous results and confirms the very high conservation of TPI during evolution. Studies of the duplicated cytosolic isozymes of phosphoglucose isomerase (EC 5.3.1.9; PGI) in Clarkia, a wild plant native to California, (Onagraceae) were continued. A competitive ELISA, using polyclonal antibodies, was devised which was able to distinguish SDS-denatured PGIs coded by the duplicate loci. In vitro dissociation and reassociation experiments revealed that the duplicated subunits differed in their efficiency of reassociation. Concordant studies of substrate affinities suggested that the suplicated PGI isozymes have diverged more in structural properties than kinetic ones. Another study using immunological procedures revealed that species of Clarkia with and without the duplication have the same levels of cytosolic PGI protein and activity relative to those of the chloroplast PGI isozyme.

                        Impacts
                        (N/A)

                        Publications


                          Progress 01/01/83 to 12/30/83

                          Outputs
                          Genetic and biochemical studies of glycolytic isozymes in plants have been extended to the plastid and cytosolic isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1). We achieved the first complete purification of both isozymes from higher plants. Both are composed of two isosubunits with approximate molecular weights of 27,000. The two isozymes, purified from lettuce, had closely similar N-terminal amino acid sequences; 9/13 positions in the two proteins had identical residues. The partial amino acid sequences showed high similarity to previously sequenced animal TPIs, indicating that the protein is very highly conserved. We were successful in synthesizing a catalytically active hybrid enzyme between subunits of the plastid and cytosolic TPIs from several vegetable species. The synthesis represents the first demonstration in plants or animals that isozymes present in different subcellular compartments have compatible subunit binding sites. Since such hybrid molecules do not form in vivo, we proposed a specific mechanism that prevenets their formation in the cell.

                          Impacts
                          (N/A)

                          Publications


                            Progress 01/01/82 to 12/30/82

                            Outputs
                            Genetic and biochemical studies of plastid and cytosolic isozymes of phosphoglucose isomerase (PGI; EC 5.3.1.9), an essential enzyme in glycolysis and gluconeogenesis, have continued. Both isozymes are specified by independent nuclear gene loci. We tested alternative models of their origin; either they represent products of a duplication of an ancestral nuclear gene, or the gene specifying the plastid PGI was once part of the genome of a prokaryotic symbiont which apparently entered the ancestral plant cell and evolved into the plastid, eventually being transferred to the nucleus. To distinguish these models, antibodies were raised against separately purified and denatured plastid and cytosolic PGIs from spinach, and the immunological similarities of these two proteins and the single PGI from a cyanobacterium were assessed by the very sensitive enzyme-linked immunosorbent assay (ELISA). We found that antibodies raised against the spinach plastid PGI cross-reacted about 10-fold more strongly with the cyanobacterial PGI than with the cytosolic PGI isozyme. Antiserum raised against the cytosolic PGI displayed weak cross-reactivity to both plastid and cyanobacterial PGIs. These results suggest that the structural genes coding the two isozymes in plants may have different phylogenetic origins and be derived from both eukaryotic and prokaryotic sources.

                            Impacts
                            (N/A)

                            Publications


                              Progress 01/01/81 to 12/30/81

                              Outputs
                              Progress has been made on the development of basic biochemical procedures that are required to identify the polypeptide precursors of the chloroplast isozyme of phosphoglucose Isomerase (EC 5.3.1.9). Antibodies have been prepared in rabbits against denatured cytosolic and chloroplast isozymes extracted and fully purified from spinach. Exact procedures have been developed for making peptide maps of cytosolic PGI subunits. Analysis of the amount of chloroplast and cytosolic PGI activity in different plant organs and at different stages of development is underway.

                              Impacts
                              (N/A)

                              Publications


                                Progress 10/01/80 to 12/30/80

                                Outputs
                                Genetic and biochemical studies have shown that many diploid species of the wildplant genus Clarkia, native to California, possess a duplication of the structural gene coding cytosolic subunits of phosphoglucoisomerase (PGI, EC 5.3.1.9), a dimeric enzyme that catalyzes an essential step in glycolysis. Plants also possess another PGI isozyme located in the chloroplast. As part of our interest in understanding the evolution of the various PGI gene loci, we have used guanidine HCl to dissociate both the chloroplast and cytosolic isozymes into their constituent subunits. Reassociation tests were then used to determine whether the subunits specified by the different genes could reassociate to form hybrid molecules, even though this never occurs in vivo. We were able to show that the chloroplast subunits from diverse higher plants including spinach and sunflower reassociate to form hybrid enzymes, and that cytosolic subunits likewise reassociate, but the chloroplast and cytosolic subunits do not form hybrid molecules. Thus, the absence of hybrid molecules composed of subunits of the two isozymes probably reflects the inability of their subunit binding sites to recognize each other.

                                Impacts
                                (N/A)

                                Publications