Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.     

Formation of plant metabolic gene clusters within dynamic chromosomal regions

In bacteria, genes with related functions often are grouped together in operons and are cotranscribed as a single polycistronic mRNA. In eukaryotes, functionally related genes generally are scattered across the genome. Notable exceptions include gene clusters for catabolic pathways in yeast, synthesis of secondary metabolites in filamentous fungi, and the major histocompatibility complex in animals. Until quite recently it was thought that gene clusters in plants were restricted to tandem duplicates (for example, arrays of leucine-rich repeat disease-resistance genes). However, operon-like clusters of coregulated nonhomologous genes are an emerging theme in plant biology, where they may be involved in the synthesis of certain defense compounds. These clusters are unlikely to have arisen by horizontal gene transfer, and the mechanisms behind their formation are poorly understood. Previously in thale cress (Arabidopsis thaliana) we identified an operon-like gene cluster that is required for the synthesis and modification of the triterpene thalianol. Here we characterize a second operon-like triterpene cluster (the marneral cluster) from A. thaliana, compare the features of these two clusters, and investigate the evolutionary events that have led to cluster formation. We conclude that common mechanisms are likely to underlie the assembly and control of operon-like gene clusters in plants.     

Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021

Sinorhizobium meliloti is an alpha-proteobacterium that forms agronomically important N(2)-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.     

Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites

Streptomyces avermitilis is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites, one of which, avermectin, is commercially important in human and veterinary medicine. The major interest in this genus Streptomyces is the diversity of its production of secondary metabolites as an industrial microorganism. A major factor in its prominence as a producer of the variety of secondary metabolites is its possession of several metabolic pathways for biosynthesis. Here we report sequence analysis of S. avermitilis, covering 99% of its genome. At least 8.7 million base pairs exist in the linear chromosome; this is the largest bacterial genome sequence, and it provides insights into the intrinsic diversity of the production of the secondary metabolites of Streptomyces. Twenty-five kinds of secondary metabolite gene clusters were found in the genome of S. avermitilis. Four of them are concerned with the biosyntheses of melanin pigments, in which two clusters encode tyrosinase and its cofactor, another two encode an ochronotic pigment derived from homogentiginic acid, and another polyketide-derived melanin. The gene clusters for carotenoid and siderophore biosyntheses are composed of seven and five genes, respectively. There are eight kinds of gene clusters for type-I polyketide compound biosyntheses, and two clusters are involved in the biosyntheses of type-II polyketide-derived compounds. Furthermore, a polyketide synthase that resembles phloroglucinol synthase was detected. Eight clusters are involved in the biosyntheses of peptide compounds that are synthesized by nonribosomal peptide synthetases. These secondary metabolite clusters are widely located in the genome but half of them are near both ends of the genome. The total length of these clusters occupies about 6.4% of the genome.     

Conservation of structural and functional domains in complement component C3 of Xenopus and mammals.

The cDNA sequence and the deduced amino acid sequence of the Mr 34,000 C-terminal fragment of Xenopus laevis complement component C3 are presented. The sequence of Xenopus C3 has 57% nucleotide identity to the corresponding sequence of human C3 and approximately 49% amino acid identity to C3 from human, mouse, and rabbit. The Xenopus C3 sequence shows clusters of high and of low similarity to the mammalian C3 sequences. One of these regions of high similarity represents the domain of mammalian C3b involved in the binding of properdin, a regulator of the alternative pathway of complement activation. It is not clear whether the other highly conserved regions are involved in binding to other C3 ligands. The Xenopus C3 sequence completely lacks the Arg-Gly-Asp sequence, which has been suggested to be the recognition site of the human complement receptor type 3 on the iC3b fragment of human C3. The Xenopus C3 gene is shown not to be linked to the Xenopus major histocompatibility complex, as is also the case in mammals. Since the gene of the related molecule C4 is MHC-linked in both mammals and Xenopus, the C3 and C4 genes may have separated before Xenopus and mammals speciated.     

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.     

Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase.

In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of cellulose, the major cell wall polymer of plants. We have characterized two cotton (Gossypium hirsutum) cDNA clones and identified one rice (Oryza sativa) cDNA that are homologs of the bacterial celA genes that encode the catalytic subunit of cellulose synthase. Three regions in the deduced amino acid sequences of the plant celA gene products are conserved with respect to the proteins encoded by bacterial celA genes. Within these conserved regions, there are four highly conserved subdomains previously suggested to be critical for catalysis and/or binding of the substrate UDP-glucose (UDP-Glc). An overexpressed DNA segment of the cotton celA1 gene encodes a polypeptide fragment that spans these domains and binds UDP-Glc, while a similar fragment having one of these domains deleted does not. The plant celA genes show little homology at the N- and C-terminal regions and also contain two internal insertions of sequence, one conserved and one hypervariable, that are not found in the bacterial gene sequences. Cotton celA1 and celA2 genes are expressed at high levels during active secondary wall cellulose synthesis in developing cotton fibers. Genomic Southern blot analyses in cotton demonstrate that celA forms a small gene family.     

High concentrations of long interspersed nuclear element sequence distinguish monoallelically expressed genes

Genes subject to monoallelic expression are expressed from only one of the two alleles either selected at random (random monoallelic genes) or in a parent-of-origin specific manner (imprinted genes). Because high densities of long interspersed nuclear element (LINE)-1 transposon sequence have been implicated in X-inactivation, we asked whether monoallelically expressed autosomal genes are also flanked by high densities of LINE-1 sequence. A statistical analysis of repeat content in the regions surrounding monoallelically and biallelically expressed genes revealed that random monoallelic genes were flanked by significantly higher densities of LINE-1 sequence, evolutionarily more recent and less truncated LINE-1 elements, fewer CpG islands, and fewer base-pairs of short interspersed nuclear elements (SINEs) sequence than biallelically expressed genes. Random monoallelic and imprinted genes were pooled and subjected to a clustering analysis algorithm, which found two clusters on the basis of aforementioned sequence characteristics. Interestingly, these clusters did not follow the random monoallelic vs. imprinted classifications. We infer that chromosomal sequence context plays a role in monoallelic gene expression and may involve the recognition of long repeats or other features. The sequence characteristics that distinguished the high-LINE-1 category were used to identify more than 1,000 additional genes from the human and mouse genomes as candidate genes for monoallelic expression.     

Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae

nifU of nitrogen-fixing bacteria is involved in the synthesis of the Fe-S cluster of nitrogenase. In a synthetic lethal screen with the mitochondrial heat shock protein (HSP)70, SSQ1, we identified a gene of Saccharomyces cerevisiae, NFU1, which encodes a protein with sequence identity to the C-terminal domain of NifU. Two other yeast genes were found to encode proteins related to the N-terminal domain of bacterial NifU. They have been designated ISU1 and ISU2. Isu1, Isu2, and Nfu1 are located in the mitochondrial matrix. ISU genes of yeast carry out an essential function, because a Deltaisu1Deltaisu2 strain is inviable. Growth of Deltanfu1Delta isu1 cells is significantly compromised, allowing assessment of the physiological roles of Nfu and Isu proteins. Mitochondria from Deltanfu1Deltaisu1 cells have decreased activity of several respiratory enzymes that contain Fe-S clusters. As a result, Deltanfu1Deltaisu1 cells grow poorly on carbon sources requiring respiration. Deltanfu1Deltaisu1 cells also accumulate abnormally high levels of iron in their mitochondria, similar to Deltassq1 cells, indicating a role for these proteins in iron metabolism. We suggest that NFU1 and ISU1 gene products play a role in iron homeostasis, perhaps in assembly, insertion, and/or repair of mitochondrial Fe-S clusters. The conservation of these protein domains in many organisms suggests that this role has been conserved throughout evolution.     

Detecting patterns of protein distribution and gene expression in silico

Most biological information is contained within gene and genome sequences. However, current methods for analyzing these data are limited primarily to the prediction of coding regions and identification of sequence similarities. We have developed a computer algorithm, CoSMoS (for context sensitive motif searches), which adds context sensitivity to sequence motif searches. CoSMoS was challenged to identify genes encoding peroxisome-associated and oleate-induced genes in the yeast Saccharomyces cerevisiae. Specifically, we searched for genes capable of encoding proteins with a type 1 or type 2 peroxisomal targeting signal and for genes containing the oleate-response element, a cis-acting element common to fatty acid-regulated genes. CoSMoS successfully identified 7 of 8 known PTS-containing peroxisomal proteins and 13 of 14 known oleate-regulated genes. More importantly, CoSMoS identified an additional 18 candidate peroxisomal proteins and 300 candidate oleate-regulated genes. Preliminary localization studies suggest that these include at least 10 previously unknown peroxisomal proteins. Phenotypic studies of selected gene disruption mutants suggests that several of these new peroxisomal proteins play roles in growth on fatty acids, one is involved in peroxisome biogenesis and at least two are required for synthesis of lysine, a heretofore unrecognized role for peroxisomes. These results expand our understanding of peroxisome content and function, demonstrate the utility of CoSMoS for context-sensitive motif scanning, and point to the benefits of improved in silico genome analysis.     

Highly conserved syntenic blocks at the vertebrate Hox loci and conserved regulatory elements within and outside Hox gene clusters

Hox genes in vertebrates are clustered, and the organization of the clusters has been highly conserved during evolution. The conservation of Hox clusters has been attributed to enhancers located within and outside the Hox clusters that are essential for the coordinated "temporal" and "spatial" expression patterns of Hox genes in developing embryos. To identify evolutionarily conserved regulatory elements within and outside the Hox clusters, we obtained contiguous sequences for the conserved syntenic blocks from the seven Hox loci in fugu and carried out a systematic search for conserved noncoding sequences (CNS) in the human, mouse, and fugu Hox loci. Our analysis has uncovered unusually large conserved syntenic blocks at the HoxA and HoxD loci. The conserved syntenic blocks at the human and mouse HoxA and HoxD loci span 5.4 Mb and 4 Mb and contain 21 and 19 genes, respectively. The corresponding regions in fugu are 16- and 12-fold smaller. A large number of CNS was identified within the Hox clusters and outside the Hox clusters spread over large regions. The CNS include previously characterized enhancers and overlap with the 5' global control regions of HoxA and HoxD clusters. Most of the CNS are likely to be control regions involved in the regulation of Hox and other genes in these loci. We propose that the regulatory elements spread across large regions on either side of Hox clusters are a major evolutionary constraint that has maintained the exceptionally long syntenic blocks at the HoxA and HoxD loci.     

Evolution of mouse immunoglobulin lambda genes.

The mouse has four C lambda and two V lambda genes. We have isolated Charon 4A clones that contain all six lambda genes from a BALB/c germ-line library. We present here the DNA sequences of the C lambda 2, C lambda 3, and C lambda 4 genes and also correct what are apparently errors in previous reports of C lambda 1 protein and DNA sequences. In addition, we have analyzed cloned DNAs by restriction mapping and electron microscopy to determine the relationships among the various lambda genes. By heteroduplex analysis, two gene clusters containing JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 show homology extending from the J regions 5' of C lambda 3/C lambda 2 to just 3' of C lambda 1/C lambda 4. Other than the region between the genes, very little homology exists in the C lambda flanking regions. In contrast, V lambda 1 and V lambda 2 genes show considerable homology extending into the 5' flanking regions. Large inverted repeats are found in the 5' flanking regions of V lambda 1 and C lambda 3, as well as in the 3' flanking regions of both C lambda gene clusters. DNA sequence divergences between the C lambda genes indicate that an ancestral JC lambda x--JC lambda g gene cluster arose at about the time of the first mammals by duplication of a primordial JC lambda gene. The data further suggest that the JC lambda x--JC lambda gene cluster duplicated after the speciation of mouse and man and subsequently diverged into the present day JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 gene clusters. C lambda 4, a pseudogene, became inactive at about the time of duplication of the ancestral JC lambda x--JC lambda y cluster. Comparison of DNA sequence divergence between the V lambda 1 and V lambda 2 genes demonstrates an anomaly. The percentage of amino acid replacement changes is approximately the same for V lambda 1/V lambda 2 as for C lambda 3/C lambda 2, implying that the ancestral V lambda gene was duplicated at the same time, and possibly together with, the JC lambda x--JC lambda y cluster. However, there are fewer silent changes than amino acid replacement changes between the V lambda 1/V lambda 2 genes, suggesting either that a selective pressure acted on the silent sites or that V lambda genes have only recently been duplicated. We also consider the possibility of a gene conversion event subsequent ot a more ancient duplication.