The evolution of the plastid chromosome in land plants: gene content, gene order, gene function

This review bridges functional and evolutionary aspects of plastid chromosome architecture in land plants and their putative ancestors. We provide an overview on the structure and composition of the plastid genome of land plants as well as the functions of its genes in an explicit phylogenetic and evolutionary context. We will discuss the architecture of land plant plastid chromosomes, including gene content and synteny across land plants. Moreover, we will explore the functions and roles of plastid encoded genes in metabolism and their evolutionary importance regarding gene retention and conservation. We suggest that the slow mode at which the plastome typically evolves is likely to be influenced by a combination of different molecular mechanisms. These include the organization of plastid genes in operons, the usually uniparental mode of plastid inheritance, the activity of highly effective repair mechanisms as well as the rarity of plastid fusion. Nevertheless, structurally rearranged plastomes can be found in several unrelated lineages (e.g. ferns, Pinaceae, multiple angiosperm families). Rearrangements and gene losses seem to correlate with an unusual mode of plastid transmission, abundance of repeats, or a heterotrophic lifestyle (parasites or myco-heterotrophs). While only a few functional gene gains and more frequent gene losses have been inferred for land plants, the plastid Ndh complex is one example of multiple independent gene losses and will be discussed in detail. Patterns of ndh-gene loss and functional analyses indicate that these losses are usually found in plant groups with a certain degree of heterotrophy, might rendering plastid encoded Ndh1 subunits dispensable.     

Owl monkey gene mapping: the assignment of gene loci for catalase, beta-globin gene cluster, HRAS1, insulin, and parathyroid hormone

Using somatic cell genetics and Southern blot hybridization, we have mapped five structural genes in the owl monkey, coding for catalase (CAT), the beta-globin gene cluster (HBBC), c-Ha-ras 1 (HRAS1), insulin (INS), and parathyroid hormone (PTH). All five loci are mapped to chromosome 19 of karyotype VI (2n = 49,50) of the owl monkey; CAT, HBBC, INS, and PTH can be assigned to chromosome 4 of karyotype V (2n = 46), while CAT and HBBC can be assigned to chromosome 2 of karyotype III (2n = 53). Using in situ hybridization, the CAT gene was precisely mapped on the mid-region and the beta-globin gene cluster on the telomeric end of chromosome 2q(K-III). Our results provide significant insight into the evolutionary history of these gene loci. While these loci are separated into at least two major segments in rodents such as the mouse, our results suggest conservation of a single chromosome arm among higher primates.     

Alternative splicing, the gene concept, and evolution

Alternative splicing allows for the production of many gene products from a single coding sequence. I introduce the concept of alternative splicing via some examples. I then discuss some current hypotheses about the explanatory role of alternative splicing, including the claim that splicing is a significant contributor to the difference in complexity between the human genome and proteosome. Hypotheses such as these bring into question our working concepts of the gene. I examine several gene concepts introduced to cope with processes such as alternative splicing. Next I introduce some hypotheses about the evolution of mechanisms alternative splicing in higher organisms. I conclude that attention to alternative splicing reveals that we adopt an attitude that developmental theorizing must inform evolutionary theorizing and vice versa.     

Protein complexity, gene duplicability and gene dispensability in the yeast genome

Using functional genomic and protein structural data we studied the effects of protein complexity (here defined as the number of subunit types in a protein) on gene dispensability and gene duplicability. We found that in terms of gene duplicability the major distinction in protein complexity is between hetero-complexes, each of which includes at least two different types of subunits (polypeptides), and homo-complexes, which include monomers and complexes that consist of only subunits of one polypeptide type. However, gene dispensability decreases only gradually as the number of subunit types in a protein complex increases. These observations suggest that the dosage balance hypothesis can explain well gene duplicability of complex proteins, but cannot completely explain the difference in dispensabilities between hetero-complex subunits. It is likely that knocking out a gene coding for a hetero-complex subunit would disrupt the function of the whole complex, so that the deletion effect on fitness would increase with protein complexity. We also found that multi-domain polypeptide genes are less dispensable but more duplicable than single-domain polypeptide genes. Duplicate genes derived from the whole genome duplication event in yeast are more dispensable (except for ribosomal protein genes) than other duplicate genes. Further, we found that subunits of the same protein complex tend to have similar expression levels and similar effects of gene deletion on fitness. Finally, we estimated that in yeast the contribution of duplicate genes to genetic robustness against null mutation is approximately 9%, smaller than previously estimated. In yeast, protein complexity may serve as a better indicator of gene dispensability than do duplicate genes.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

The novel exon, exon T, of the human progesterone receptor gene and the genomic organization of the gene

Recently, we have cloned the novel isoform of the progesterone receptor (PR) cDNA (PR isoform S cDNA) from the human testicular cDNA library. The isoform S cDNA consists of the novel exon (termed the exon S of the PR gene) and the exons 4-8 of the PR gene. In order to investigate the existence of the other isoform of the human PR cDNA, the human testicular cDNA library was screened by the exons 4-8 corresponding sequence of the human PR cDNA in the present study. As a result, we have identified a novel isoform of the PR cDNA (termed the PR isoform T cDNA (PR-T cDNA)), which consisted of a previously unidentified 5'-sequence and the exons 4-8 of the PR gene. The structure of this isoform T cDNA is essentially similar to that of the isoform S cDNA. By the genomic cloning, the 5'-sequence of the PR isoform T mRNA was demonstrated to originate from a novel independent exon, exon T, which was located in the 5'-upstream region of the exon S.     

Studies on the mutator gene, mutT of Escherichia coli. Molecular cloning of the gene, purification of the gene product, and identification of a novel nucleoside triphosphatase

The mutator gene, mutT, has been cloned into an expression vector and overproduced in Escherichia coli. The gene product has been purified to over 90% homogeneity as judged by gel electrophoresis and amino acid analysis. The amino acid composition of the protein and the sequence of the 20 amino acids of the N-terminal region agree well with the nucleotide sequence of the gene reported by Akiyama et al. (Akiyama, M., Horiuchi, T., and Sekiguchi, M. (1987) Mol. Gen. Genet. 206, 9-16) and indicate that the first of the potential initiation codons (position 164) of the open reading frame in the PvuII fragment carrying the mutT gene is the site of initiation of translation of the 15,000-Da polypeptide. A novel nucleoside triphosphatase activity which has a preference for dGTP is associated with the purified protein, and preliminary experiments are consistent with the notion that the mutT gene product is the enzyme responsible for this activity.     

Mitotic gene conversion, reciprocal recombination and gene replacement at the benA, beta-tubulin, locus of Aspergillus nidulans

Summary We have developed a procedure for determining the rates of mitotic recombination of an interrupted duplication created by integration of transforming plasmid sequences at the benA, beta-tubulin, locus of Aspergillus nidulans. Transformation of a strain carrying a benomyl-resistant benA allele with plasmid AIpGM4, which carries the wild-type benA allele and the pyr4 (orotidine-5-phosphate decarboxylase) gene of Neurospora crassa, creates an interrupted duplication with plasmid sequences flanked by two benA alleles, one wild type and one benomyl resdistant. Such transformants will not grow in the presence of high levels of benomyl. Mitotic recombination causes the loss of the wild-type benA allele or conversion of the wild-type to the mutant allele resulting in nuclei carrying only the benomylresistant allele. Conidia containing such nuclei can be selected on media with high benomyl allowing easy quantitation of mitotic recombination. We found that the rate of recombination giving rise to benomyl-resistant conidia was 4.6×10^-4. Reciprocal recombination leading to benomyl-resistant conidia lacking plasmid sequences occurred at a rate of 2.0×10^-4 and gene conversion leading to benomylresistant conidia occurred at a rate of 2.6×10^-4. We selected for reciprocal recombination leading to loss of pyr4 sequences on 5-fluoro-orotic acid and used this selection for two-step gene replacement of a mutant benA allele with the wild-type allele.     

Is the egg size determining gene,Esd, on the W chromosome identical with the sex-linked giant egg gene,Ge, in the silkworm?

The presence of the egg size-determining (Esd) gene, which acts as a quantitative gene, on the W chromosome of the silkworm was revealed in a previous study by using two types of triploid females, ZZW and ZWW, (Kawamura, Genetica 76: 195–201). The females with the sex-linked giant egg (Ge) gene deposit eggs as large as those laid by tetraploids. If the Ge mutant is induced by translocation of a fragment of the W carrying Esd onto the Z by chance, the egg size increase in the Ge strain and in tetraploids may be easily explained by the double dose of Esd. The measurement of the length of the Z-W bivalent in oocytes showed that the Z of the Ge strain was much longer than that of the other strains which do not carry the Ge gene. The result suggests that the Ge gene is identical with the Esd on the W chromosome of the silkworm.     

Sequence and characterization of pvuIIR, the PvuII endonuclease gene, and of pvuIIC, its regulatory gene.

An open reading frame partially overlaps pvuIIR, and genetic evidence implies that this open reading frame, named pvuIIC, specifies a positive regulator of pvuIIR (T. Tao, J. C. Bourne, and R. M. Blumenthal, J. Bacteriol. 173:1367-1375, 1991). Inducible constructs of pvuIIC produced a protein of the expected size. The site of C.PvuII action appears to lie within pvuIIC itself; thus, pvuIIC may be a self-contained regulatory cassette.     

The tkNeo gene, but not the pgkPuro gene, can influence the ability of the beta-globin LCR to enhance and confer position-independent expression onto the beta-globin gene

Whether drug-selectable genes can influence expression of the beta-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the beta-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the beta-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3' end of the beta-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3' end of the beta-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the beta-globin gene linked to its LCR. The findings could partly explain why data on beta-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.     

KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene

The yeast KAR2 gene was isolated by complementation of a mutation that blocks nuclear fusion. The predicted KAR2 protein sequence is most homologous to mammalian BiP/GRP78 and has several structural features in common with it: a functional secretory signal sequence, a yeast endoplasmic reticulum retention signal (HDEL) at the carboxyl terminus, and the absence of potential N-linked glycosylation sites. Moreover KAR2 is regulated like BiP/GRP78: the level of mRNA is increased by drug treatments and mutations that cause accumulation of secretory precursors in the endoplasmic reticulum. However, unlike BiP/GRP78, KAR2 is also regulated by heat shock. Deletion of the KAR2 gene generated a recessive lethal mutation, showing that BiP/GRP78 function is required for cell viability.     

Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes.

Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species by an activation-decarboxylation reaction which yields formate and CO2. oxc, the gene encoding the oxalic acid-degrading enzyme oxalyl-coenzyme A decarboxylase, was cloned from the bacterium Oxalobacter formigenes. The DNA sequence revealed a single open reading frame of 1,704 bp capable of encoding a 568-amino-acid protein with a molecular weight of 60,691. The identification of a presumed promoter region and a rho-independent termination sequence indicates that this gene is not part of a polycistronic operon. A PCR fragment encoding the open reading frame, when overexpressed in Escherichia coli, produced a product which cross-reacted antigenically with native enzyme on Western blots (immunoblots), appeared to form homodimers spontaneously, and exhibited enzymatic activity similar to that of the purified native enzyme.