Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

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The DNA polymerase gene from the Archaea In 1985, a species of extreme thermophile was isolated from a submarine thermal vent near Naples, Italy (1). This organism, Thermococcus litoralis, can be cultured at up to 980C and contains a heat-stable DNA polymerase that we call Vent DNA polymerase (New England Biolabs). This paper describes the cloning, sequencing, and expression of the Vent DNA polymerase gene ¶ and the finding of two intervening sequences (IVSs) that make up 55% of the polymerase gene, one of which, IVS2, encodes the I-Tli I (I, intron) endonuclease. To our knowledge, this is the first report of introns in protein coding genes of Archaea or eubacteria, although introns have been found in protein coding genes of eubacteriophage (2). Previously described Archaea or eubacterial introns are mainly pre-tRNA or self-splicing introns in stable .Introns often contain open reading frames (ORFs) that are in-frame with either the 5' or 3' exon, but not with both exons. An intron in the Saccharomyces cerevisiae TFP1 gene forms a single ORF with the surrounding exons; the authors proposed (6, 7) that this intron is spliced at the protein, not the mRNA, level. In the present study, we describe two introns that form a single ORF with the surrounding exons. Furthermore, we present evidence indicating that the Vent DNA polymerase IVSs are removed either by protein splicing or by RNA splicing that requires I-Tli I as a maturase.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modifled DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB+-dependent The locus known as mcrB was one of the first restriction systems to be discovered (33), by virtue of its action on special variants of T-even bacteriophage that incorporate 5-hydroxymethylcytosine (hm`C) into their DNA without further modification (see reference 50 for a review). This locus, formerly known as rglB (or r2,4) (48), was rediscovered because of difficulties encountered in cloning the genes for site-specific modification methylases associated with type II restriction-modification systems (7,26,40,49). In addition to hm5C-DNA, many but not all sequences methylated by site-specific cytosine modification methylases are restricted by the system in vivo, and the consensus recognition sequence 5'GmC was proposed (49). McrB is thus a sequence-specific, modification-requiring restriction system. We show here that the mcrB locus described above actually comprises two genes and that both are required for restriction of most the sequences previously characterized as sensitive. Thus, we will refer to the complete system as the McrBC system.The genes encoding the system are contained within the immigration control region of the Escherichia coli K-12 genome. Three restriction systems are encoded within 14 kilobases (kb) here (48). The well-studied hsdRMS locus (20, 31, 55) encodes the multisubunit type I system EcoK, which recognizes a seven-base sequence and cleaves the target when the sequence is not modified. The other two systems are the flanking loci mcrBC, described above, and mrr, which mediates site-specific restriction of adenine-modified DNA (22). The sequence organization of this region, judged by Southern blot analysis of chromosomal DNA, is highly variable in enteric bacteria (12), both in the hsd genes specifically and in the flanking sequences. Sequence analysis presented here is consistent with recent acquisition of the mcrBC genes by E. coli, possible accounting for some of the observed variability. * Corresponding author.At the molecular level, restriction systems consist of sequence-specific double-stranded endonucleases, usually accompanied by a sequence-specific modification methylase. So far, four classes of endonucleases have been described. The simplest are the type II enzymes, in which the endonuclease and protective methylase activities reside in separate enzymes. These endonucleases typically act as dimers of identical subunits and require only Mg2" for activity (38).One group of type II isoschizomers, typified by DpnI, recognizes a modified site (28), as McrBC appears to do. In contrast, type I and type III enzymes have separate specificity subunits that recognize the DNA site and require ATP in a...

The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) DNA (39,41,44,45). These two systems were initially called rglA and rglB because they restricted glucoseless, 5-hydroxylmethyl cytosine-containing DNA, present in many T-even phage (42). One system (rglB) was found to produce an endonuclease activity (16). These restriction systems differ from the classically recognized ones since they require modified DNA as the substrate for their action rather than using modification for selfprotection.An additional methylation-specific restriction system of E. coli, Mrr, was described by Heitman and Model (20) and was shown to interfere with the maintenance of certain N6-adenine methylases. The HhaII and PstI N6-adenine methylase genes, when maintained in several E. coli K-12 strains, produced DNA damage as evidenced by induction of the SOS DNA repair response (20). They also demonstrated that several cytosine methylases induced the SOS response. Transposon insertion mapping and Southern blotting were used to position mrr on the E. coli chromosome at 98.5 min.

The two divergently transcribed H2A-H2B gene pairs in yeast are differentially regulated as a function of the copy number of histone genes. Transcription of an HTA2-1acZ reporter gene is independent of histone gene copy number. Transcription of an HTAI-lacZ gene can be repressed or derepressed, depending on the number of HTA plus HTB genes in cells. Regulation by histone gene dosage is dependent on a negative site in the HTA1-HTB1 promoter and the products of regulatory genes that act through this site. The level of H2A plus H2B protein in the cell may signal the response to histone gene copy number, suggesting that transcription of the HTA1-HTB1 locus can be autogenously regulated. This phenomenon may be used, in part, to maintain the balanced synthesis of histones, a critical parameter in nucleosome assembly.

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The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.