The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.
A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissueactivating peptide III (two human platelet a-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variait of platelet factor 4, designated PF4varl. The gene for PF4varl consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4varl had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4varl genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4varl. Rat PF4 and human PF4varl genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.
A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rn7J and rrnWin Bacilus subtilis has been cloned and sequenced. This cluster contains the genes for tRNAuv'C, tRNA JU, tRNAjuSuu tRNAu`ZG, tRNAaC, tRNAUZA%, tRNA G, tRNAuGG9, and tRNAAuC. The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnl and rrnH, and of the 5'-end oftrnB, a cluster of21 tRNA genes found immediately 3' to rrnB. Neither the tRNAu'5ZG gene nor its product has been found previously in B. subtUis. With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B. subtUis. These known genes account for almost all of the tRNA hybridizing restriction fragments of the B. subtilis genome. The 60 known tRNA genes ofB. subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli. This may indicate that B. subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum.The tRNA genes of Bacillus subtilis are highly clustered. Groups of 21, 16, 6, 4, and 2 genes have been reported (5,9,12,24,25,28). All except the cluster of four tRNA genes (28) are closely linked to rRNA operons. Ribosomal RNA operons rrnO and rrnA have genes for tRNAIl' and tRNAAla between the 16S and 23S genes, similar to the organization of Escherichia coli rRNA operons (9, 12). The other known clusters are immediately 3' to rRNA operons. These tRNA groups are named after the preceding rRNA operon (13). The 21-tRNA gene cluster, trnB, immediately follows the rRNA operon rmB at 2800 on the chromosome (3, 4). The 16-tRNA gene cluster also immediately follows an rRNA operon, but it has not yet been mapped. Transcriptional analysis indicates that although both trnB and the 16-tRNA gene cluster have internal promoters to transcribe the tRNA genes, they can also be transcribed from the upstream rRNA promoter, as there are no terminators between the rRNA and tRNA genes (21-23).
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