Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli

A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.     

Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta

Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537-bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.     

Rational genomics I: antisense open reading frames and codon bias in short-chain oxido reductase enzymes and the evolution of the genetic code

The short-chain oxidoreductase (SCOR) family of enzymes includes over 6000 members, extending from bacteria and archaea to humans. Nucleic acid sequence analysis reveals that significant numbers of these genes are remarkably free of stopcodons in reading frames other than the coding frame, including those on the antisense strand. The genes from this subset also use almost entirely the GC-rich half of the 64 codons. Analysis of a million hypothetical genes having random nucleotide composition shows that the percentage of SCOR genes having multiple open reading frames exceeds random by a factor of as much as 1 x 10(6). Nevertheless, screening the content of the SWISS-PROT TrEMBL database reveals that 15% of all genes contain multiple open reading frames. The SCOR genes having multiple open reading frames and a GC-rich coding bias exhibit a similar GC bias in the nucleotide triple composition of their DNA. This bias is not correlated with the GC content of the species in which the SCOR genes are found. One possible explanation for the conservation of multiple open reading frames and extreme bias in nucleic acid composition in the family of Rossman folds is that the primordial member of this family was encoded early using only very stable GC-rich DNA and that evolution proceeded with extremely limited introduction of any codons having two or more adenine or thymine nucleotides. These and other data suggest that the SCOR family of enzymes may even have diverged from a common ancestor before most of the AT-rich half of the genetic code was fully defined.     

Constraint on di-nucleotides by codon usage bias in bacterial genomes

It has been reported earlier that the relative di-nucleotide frequency (RDF) in different parts of a genome is similar while the frequency is variable among different genomes. So RDF is termed as genome signature in bacteria. It is not known if the constancy in RDF is governed by genome wide mutational bias or by selection. Here we did comparative analysis of RDF between the inter-genic and the coding sequences in seventeen bacterial genomes, whose gene expression data was available. The constraint on di-nucleotides was found to be higher in the coding sequences than that in the inter-genic regions and the constraint at the 2nd codon position was more than that in the 3rd position within a genome. Further analysis revealed that the constraint on di-nucleotides at the 2nd codon position is greater in the high expression genes (HEG) than that in the whole genomes as well as in the low expression genes (LEG). We analyzed RDF at the 2nd and the 3rd codon positions in simulated coding sequences that were computationally generated by keeping the codon usage bias (CUB) according to genome G+C composition and the sequence of amino acids unaltered. In the simulated coding sequences, the constraint observed was significantly low and no significant difference was observed between the HEG and the LEG in terms of di-nucleotide constraint. This indicated that the greater constraint on di-nucleotides in the HEG was due to the stronger selection on CUB in these genes in comparison to the LEG within a genome. Further, we did comparative analyses of the RDF in the HEG rpoB and rpoC of 199 bacteria, which revealed a common pattern of constraints on di-nucleotides at the 2nd codon position across these bacteria. To validate the role of CUB on di-nucleotide constraint, we analyzed RDF at the 2nd and the 3rd codon positions in simulated rpoB/rpoC sequences. The analysis revealed that selection on CUB is an important attribute for the constraint on di-nucleotides at these positions in bacterial genomes. We believe that this study has come with major findings of the role of CUB on di-nucleotide constraint in bacterial genomes.     

An ATP hydrolysis sensor in the DNA packaging motor from bacteriophage T4 suggests an inchworm-type translocation mechanism

Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.     

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.     

Bioinformatic analysis of the Acinetobacter baumannii phage AB1 genome

As one of the pathogens of hospital-acquired infections, Acinetobacter baumannii poses great challenges to the public health. A. baumannii phage could be an effective way to fight multi-resistant A. baumannii. Here, we completed the whole genome sequencing of the complete genome of A. baumannii phage AB1, which consists of 45,159bp and is a double-stranded DNA molecule with an average GC content of 37.7%. The genome encodes one tRNA gene and 85 open reading frames (ORFs) and the average size of the ORF is 531bp in length. Among 85 ORFs, only 14 have been identified to share significant sequence similarities to the genes with known functions, while 28 are similar in sequence to the genes with function-unknown genes in the database and 43 ORFs are uniquely present in the phage AB1 genome. Fourteen function-assigned genes with putative functions include five phage structure proteins, an RNA polymerase, a big sub-unit and a small sub-unit of a terminase, a methylase and a recombinase and the proteins involved in DNA replication and so on. Multiple sequence alignment was conducted among those homologous proteins and the phylogenetic trees were reconstructed to analyze the evolutionary courses of these essential genes. From comparative genomics analysis, it turned out clearly that the frame of the phage genome mainly consisted of genes from Xanthomonas phages, Burkholderia ambifaria phages and Enterobacteria phages and while it comprises genes of its host A. baumannii only sporadically. The mosaic feature of the phage genome suggested that the horizontal gene transfer occurred among the phage genomes and between the phages and the host bacterium genomes. Analyzing the genome sequences of the phages should lay sound foundation to investigate how phages adapt to the environment and infect their hosts, and even help to facilitate the development of biological agents to deal with pathogenic bacteria.     

Factors influencing the synonymous codon and amino acid usage bias in AT-rich Pseudomonas aeruginosa phage PhiKZ

To reveal how the AT-rich genome of bacteriophage PhiKZ has been shaped in order to carryout its growth in the GC-rich host Pseudomonas aeruginosa,synonymous codon and amino acid usage bias ofPhiKZ was investigated and the data were compared with that of P.aeruginosa.It was found that synonymouscodon and amino acid usage of PhiKZ was distinct from that of P.aeruginosa.In contrast to P.aeruginosa,the third codon position of the synonymous codons of PhiKZ carries mostly A or T base;codon usage biasin PhiKZ is dictated mainly by mutational bias and,to a lesser extent,by translational selection.A clusteranalysis of the relative synonymous codon usage values of 16 myoviruses including PhiKZ shows that PhiKZis evolutionary much closer to Escherickia coli phage T4.Further analysis reveals that the three factors ofmean molecular weight,aromaticity and cysteine content are mostly responsible for the variation of aminoacid usage in PhiKZ proteins,whereas amino acid usage of P.aeruginosa proteins is mainly governed bygrand average of hydropathicity,aromaticity and cysteine content.Based on these observations,we suggestthat codons of the phage-like PhiKZ have evolved to preferentially incorporate the smaller amino acid residuesinto their proteins during translation,thereby economizing the cost of its development in GC-rich P.aeruginosa.     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.     

Nuclear-encoded chloroplast RNA polymerase sigma factor SIG2 activates chloroplast-encoded phycobilisome genes in a red alga, Cyanidioschyzon merolae

The phycobilisome (PBS) is a photosynthetic light-harvesting complex in red algae, whose structural genes are separately encoded by both the nuclear and chloroplast genomes. While the expression of PBS genes in both genomes is responsive to environmental changes to modulate light-harvesting efficiency, little is known about how gene expression of the two genomes is coordinated. In this study, we focused on the four nuclear-encoded chloroplast sigma factors to understand aspects of this coordination, and found that SIG2 directs the expression of chloroplast PBS genes in the red alga Cyanidioschyzon merolae.     

A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105

Penicillin acylase (PA) is an industrial enzyme that is used to convert penicillin G into a precursor for semisynthetic penicillins. We have cloned a segment of DNA that codes for the two subunits required for PA activity. We also report the nucleotide sequence of a DNA fragment that codes for (i) the small subunit, (ii) the N-terminal region of the large subunit and (iii) a putative connecting peptide. These results confirm the existence of a common precursor for both peptides.     

Differential expression of boule and dazl in adult germ cells of the Asian seabass

Fertility genes boule and dazl constitute the evolutionarily conserved DAZ (Deleted in AZoospermia) family of RNA binding proteins essential for germline development across animal phyla. Here we report the cloning and expression analysis of boule and dazl from the Asian seabass (Lates calcarifer), a marine fish that undergoes sequential male-to-female sex reversal. Molecular cloning and sequence comparison led to the identification of boule and dazl cDNAs. RT-PCR analysis showed that both boule and dazl RNAs were restricted to the gonads among adult organs examined. Chromogenic in situ hybridization revealed germ cell-specific expression for both boule and dazl in female and male adults. Importantly, distinct differences were found between boule and dazl in terms of temporospatial expression and subcellular distribution. The boule RNA was abundant in late gametogenic cells except sperm. Interestingly, dazl expression increases in early oocytes and concentrates in a perinuclear speckle that appears to develop ultimately into the Balbiani body in advanced oocytes. The dazl RNA was found to be abundant in spermatocytes but hardly detectable in sperm. These data demonstrate that boule and dazl are germ cell markers in the adult Asian seabass, and that bisexual germline-specific expression has been conserved for boule and dazl in fish.     

Cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus)

The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia.     

Sequence comparison of human and murine erythrocyte alpha-spectrin cDNA

The results of hybridization analyses using cDNA probes for mouse and human alpha-spectrin mRNA indicate that a single gene encodes the alpha-subunit of erythrocyte spectrin. Sequencing of the cDNA clones showed that they code for 370 amino acids (aa) covering three repeat domains close to the C terminus of alpha-spectrin. The cloned cDNAs will now permit the isolation of the alpha-spectrin gene and should lead to the characterization of the genetic aspects in human hereditary anemias in which alpha-spectrin has been characterized as the site of the molecular defect.     

Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli

A gene coding for xylanase synthesis in Bacillus subtilis was isolated by direct shotgun cloning using Escherichia coli as a host. Following partial digestion of B. subtilis chromosomal DNA with PstI or EcoRI restriction enzymes, fragments ranging from 3 to 7 kb were introduced into the PstI or EcoRI sites of pBR325. Transformed colonies having lost either the ampicillin or chloramphenicol resistance markers were screened directly on 1% xylan plates. Out of 8000 transformants, ten xylanase-positive clones were identified by the clearing zone around lysozyme-treated colonies. Further characterization of one of the clones showed that the xylanase gene was present in a 3.9-kb insert within the PstI site of the plasmid pBR325. Retransformation of E. coli strain with the xylanase-positive hybrid plasmid pRH271 showed 100% transformation to xylanase production. The intracellular xylanase produced by the transformed E. coli was purified by ion exchange and gel permeation chromatography. The electrophoretic mobility of the purified xylanase indicated an Mr of 22 000.