Computational identification and characterization of novel genes from legumes

The Fabaceae, the third largest family of plants and the source of many crops, has been the target of many genomic studies. Currently, only the grasses surpass the legumes for the number of publicly available expressed sequence tags (ESTs). The quantity of sequences from diverse plants enables the use of computational approaches to identify novel genes in specific taxa. We used BLAST algorithms to compare unigene sets from Medicago truncatula, Lotus japonicus, and soybean (Glycine max and Glycine soja) to nonlegume unigene sets, to GenBank's nonredundant and EST databases, and to the genomic sequences of rice (Oryza sativa) and Arabidopsis. As a working definition, putatively legume-specific genes had no sequence homology, below a specified threshold, to publicly available sequences of nonlegumes. Using this approach, 2,525 legume-specific EST contigs were identified, of which less than three percent had clear homology to previously characterized legume genes. As a first step toward predicting function, related sequences were clustered to build motifs that could be searched against protein databases. Three families of interest were more deeply characterized: F-box related proteins, Pro-rich proteins, and Cys cluster proteins (CCPs). Of particular interest were the >300 CCPs, primarily from nodules or seeds, with predicted similarity to defensins. Motif searching also identified several previously unknown CCP-like open reading frames in Arabidopsis. Evolutionary analyses of the genomic sequences of several CCPs in M. truncatula suggest that this family has evolved by local duplications and divergent selection.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting expression of nonregulated genes. The three genes code for poly(A)⁺RNAs that begin to accumulate at different times during conidiation. The brlA-and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.     

A physical map of the human PI and AACT genes

We have used probes from the human genes PI, PIL, and AACT (alpha 1-antitrypsin, alpha 1-antitrypsin-related sequence, and alpha 1-antichymotrypsin) to make a pulsed-field map of the surrounding region of 14q31-32. We have discovered that the PI-PIL gene cluster is only 220 kb away from the AACT gene and that it is orientated in the opposite direction. The comparatively short distance between the genes comes as a surprise given previous estimates of the level of genetic recombination between them.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans.

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA, and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting the expression of nonregulated genes. The three genes code for poly(A)+ RNAs that begin to accumulate at different times during conidiation. The brlA- and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.     

A physical map of the human pseudoautosomal region.

A physical map of the human pseudoautosomal region has been constructed using pulsed field gel electrophoresis and the infrequently cutting restriction enzymes BssHIII, EagI, SstII, NotI, MluI and NruI. This map extends 2.3 Mbp from the telomere to sex-chromosome-specific DNA, includes at least seven CpG islands and locates four genetically mapped loci. Five of the CpG islands are organized into two clusters. One cluster is adjacent to the telomere, the other extends into sex-chromosome-specific DNA. There is congruence between the genetic and physical maps which implies that the frequency of recombination is approximately uniform throughout the DNA.     

A deletion map of the WAGR region on chromosome 11.

The WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) region has been assigned to chromosome 11p13 on the basis of overlapping constitutional deletions found in affected individuals. We have utilized 31 DNA probes which map to the WAGR deletion region, together with six reference loci and 13 WAGR-related deletions, to subdivide this area into 16 intervals. Specific intervals have been correlated with phenotypic features, leading to the identification of individual subregions for the aniridia and Wilms tumor loci. Delineation, by specific probes, of multiple intervals above and below the critical region and of five intervals within the overlap area provides a framework map for molecular characterization of WAGR gene loci and of deletion boundary regions.     

Isolation and characterization of three novel serine protease genes from Xenopus laevis

Three novel cDNAs encoding serine proteases, that may play a role in early vertebrate development, have been identified from Xenopus laevis. These Xenopus cDNAs encode trypsin-like serine proteases and are designated Xenopus embryonic serine protease (Xesp)-1, Xesp-2, and XMT-SP1, a homolog of human MT-SP1. Xesp-1 is likely to be a secreted protein that functions in the extracellular space. Xesp-2 and XMP-SP1 are likely to be type II membrane proteases with multidomain structures. Xesp-2 has eight low density lipoprotein receptor (LDLR) domains and one scavenger receptor cysteine-rich (SRCR) domain, and XMT-SP1 has four LDLR domains and two CUB domains. The temporal expressions of these serine protease genes show distinct and characteristic patterns during embryogenesis, and they are differently distributed in adult tissues. Overexpression of Xesp-1 caused no significant defect in embryonic development, but overexpression of Xesp-2 or XMT-SP1 caused defective gastrulation or apoptosis, respectively. These results suggest that these proteases may play important roles during early Xenopus development, such as regulation of cell movement in gastrulae.     

Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza

Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.     

Petite deletion map of the mitochondrial oxi3 region in Saccharomyces cerevisiae.

Summary Fifty eight mitochondrial mutants (p ⁺ mit^- mutants), all deficient in cytochrome oxidase activity and previously assigned to the genetic region oxi3 on the mitochondrial DNA, were mapped by the method of petite deletion mapping.This procedure resulted in the identification of at least twenty one different classes of oxi3 mutants, which could be arranged in a linear order.Moreover, it provided a set of twenty three p ^- petite mutants, each containing a differentially deleted mit DNA segment included in the oxi3 region. The two sets of mutants, p ⁺ oxi3 ^- and p ^- oxi3 ⁺, will be of interest for a further genetic and physical analysis of this mitochondrial DNA segment which spans over about ten thousand base pairs and controls the subunit I of cytochrome oxidase.     

A genetic,deletion, physical,and human homology map of the long fin region on zebrafish linkage group 2

Mutation of the gene long fin causes overgrowth of zebrafish fins. Thus, molecular identification of the gene long fin may reveal the mechanisms involved in normal growth control. We have therefore developed genetic and physical maps of the corresponding region on linkage group 2 (LG2). A single deletion allele (lof(jg)(61)) of the long fin gene was also generated. Examination of this deletion for the presence or absence of ESTs independently mapped to LG2 revealed a contiguous deletion of SSLP, STS, and gene-based markers spanning a physical distance of approximately 500 kb, including at least 10 zebrafish genes. Human orthologs of the zebrafish genes in the long fin region were identified and revealed two synteny segments from human chromosome 1 (Hsa1) and Hsa19. Homology searching for additional genes linked to the human orthologs revealed one additional gene in the long fin deletion region. Thus, our development of the genetic, physical, deletion, and human homology maps of the long fin region provides one of the first high-resolution comparisons of a zebrafish region with a homologous human region, and facilitates the molecular identification of the long fin gene.