Gene content and density in banana ( Musa acuminata) as revealed by genomic sequencing of BAC clones.

The complete sequence of Musa acuminata bacterial artificial chromosome (BAC) clones is presented and, consequently, the first analysis of the banana genome organization. One clone (MuH9) is 82,723 bp long with an overall G+C content of 38.2%. Twelve putative protein-coding sequences were identified, representing a gene density of one per 6.9 kb, which is slightly less than that previously reported for Arabidopsis but similar to rice. One coding sequence was identified as a partial M. acuminata malate synthase, while the remaining sequences showed a similarity to predicted or hypothetical proteins identified in genome sequence data. A second BAC clone (MuG9) is 73,268 bp long with an overall G+C content of 38.5%. Only seven putative coding regions were discovered, representing a gene density of only one gene per 10.5 kb, which is strikingly lower than that of the first BAC. One coding sequence showed significant homology to the soybean ribonucleotide reductase (large subunit). A transition point between coding regions and repeated sequences was found at approximately 45 kb, separating the coding upstream BAC end from its downstream end that mainly contained transposon-like sequences and regions similar to known repetitive sequences of M. acuminata. This gene organization resembles Gramineae genome sequences, where genes are clustered in gene-rich regions separated by gene-poor DNA containing abundant transposons.

Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

Orthologous DNA sequence variation among 5S ribosomal RNA gene spacer sequences on homoeologous chromosomes 1B, 1D, and 1R of wheat and rye.

5S ribosomal gene spacer sequences from the short-spacer arrays of wheat and rye were isolated by PCR. The 29 new DNA sequences displayed noticeable heterogeneity at scattered positions. Nevertheless, based on shared DNA sequence polymorphisms, sequence alignment clearly classified the sequences into three groups. Group-specific primer sets were designed to allow chromosomal assignment by PCR on nullitetrasomic wheat stocks, as well as on wheat-rye translocation and addition lines. The three groups were assigned to orthologous loci 5S-Rrna-B1, 5S-Rrna-D1, and 5S-Rrna-R1 on homoeologous chromosomes 1B, 1D, and 1R, respectively. Hence, group-specific DNA sequence variation could be related to fixed orthologous DNA sequence variation between 5S rRNA multigene families on the homoeologous group 1 chromosomes. In addition, members of the three groups showed fixed orthologous length polymorphism. Four sequenced 5S-Rrna-B1 units, however, had a duplication in the gene encoding region and are probably representatives of a nontranscribed subfamily of 5S rDNA repeating units. The observed chromosome-specific polymorphisms among sequences belonging to a multigene family with thousands of copies suggests that this type of polymorphism may exist in many genes and gene families in polyploid wheats. The implication of this finding in relation to the construction of molecular tools for wheat-genome analysis and manipulation is discussed.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

Insertional re-activation of a chloramphenicol acetyltransferase misfolding mutant protein.

The deletion of nine residues from the C-terminus of the bacterial chloramphenicol acetyltransferase (CAT) results in deposition of the mutant protein in cytoplasmic inclusion bodies and loss of chloramphenicol resistance in Escherichia coli. This folding defect is relieved by C-terminal fusion of the polypeptide with as few as two residues. Based on these observations, efficient positive selection for the cloning of DNA fragments has been demonstrated. The cloning vector encodes a C-terminally truncated CAT protein. Restriction sites in front of the stop codon allow the insertion of target DNA, resulting in the production of properly folded CAT fusion proteins and regained chloramphenicol resistance. The positive selection of recombinants is accomplished by growth of transformants on chloramphenicol-containing agar plates. The method appears particularly convenient for the cloning of DNA fragments amplified by the PCR because minimal information to restore CAT folding can be included in the primers. The cloning of random sequences shows that the folding defect can be relieved by fusion to a wide variety of peptides, providing great flexibility to the positive selection system. This vector may also contribute to the determination of the role of the C-terminus in CAT folding.

Twelve open reading frames revealed in the 23.6 kb segment flanking the centromere on the Saccharomyces cerevisiae chromosome XIV right arm.

The nucleotide sequence of 23.6 kb of the right arm of chromosome XIV is described, starting from the centromeric region. Both strands were sequenced with an average redundancy of 4.87 per base pair. The overall G+C content is 38.8% (42.5% for putative coding regions versus 29.4% for non-coding regions). Twelve open reading frames (ORFs) greater than 100 amino acids were detected. Codon frequencies of the twelve ORFs agree with codon usage in Saccharomyces cerevisiae and all show the characteristics of low level expressed genes. Five ORFs (N2019, N2029, N2031, N2048 and N2050) are encoded by previously sequenced genes (the mitochondrial citrate synthase gene, FUN34, RPC34, PRP2 and URK1, respectively). ORF N2052 shows the characteristics of a transmembrane protein. Other elements in this region are a tRNA(Pro) gene, a tRNA(Asn) gene, a tau 34 and a truncated delta 34 element. Nucleotide sequence comparison results in relocation of the SIS1 gene to the left arm of the chromosome as confirmed by colinearity analysis.

Nucleotide sequence analysis of an 8887 bp region of the left arm of yeast chromosome XIV, encompassing the centromere sequence.

The nucleotide sequencing of 8887 bp of the left arm of chromosome XIV is described. The sequence includes the centromeric region. Both strands were sequenced with an average redundancy of 5.09 per base pair. The overall G+C content is 37.3% (39.2% for putative coding regions versus 32.5% for non-coding regions). Six open reading frames (ORFs) greater than 100 amino acids were detected, all of which are completely confined to the 8.9 kbp region. Codon frequencies of the six ORFs agree with codon usage in Saccharomyces cerevisiae and all show the characteristics of low-level expressed genes. Comparison of the translated sequences with protein sequences in data bases suggests the presence of two ORFs (N2014 and N2007) encoding ribosomal proteins, the latter of which is the previously sequenced MRP7 gene. Another ORF (N2012) could encode a membrane-associated protein since it contains secretory signal sequence and two presumed transmembrane helices. This protein might be involved in mitochondrial energy transfer. ORF N2016 is immediately adjacent to the centromere, suggesting that it corresponds to the SPO1 gene, which is very tightly linked to the centromere at the left arm side of chromosome XIV (Mortimer et al., 1989).

Two Streptomyces lividans 66 transfer RNA genes with anticodons corresponding to serine (AGC) and arginine (CGU) codons.

From the nucleotide sequence of a 2110-bp Streptomyces lividans 66 DNA fragment two transfer RNA genes were identified: tRNA(Ser) (AGC) and tRNA(Arg) (CGU). These tRNA genes are transcribed from the same DNA strand and both are preceded by a putative promoter structure. They are separated by an intergenic region of 191 bp. Like most Streptomyces tRNA genes described so far, they do not encode the 3' terminal CCA of mature tRNAs. Both genes are followed by extensive inverted repeats, which could serve as transcriptional terminator signals. Remarkably, these hairpin structures share an identical 9 base pair stretch (5'-GAAGCCCCG-3'). Furthermore, the tRNA(Arg) region is followed by two potential open reading frames, which are encoded on complementary strands and have 3' overlapping ends. The gene for tRNA(Arg) (CGU) is the first Streptomyces tRNA gene described so far which encodes the translation of a codon with uridine at the third (wobble) position.