A pair of Bacillus subtilis ribosomal protein genes mapping outside the principal ribosomal protein cluster.

Before now, the only ribosomal protein gene loci to be identified in Bacillus subtilis map within the principal ribosomal protein gene cluster at about 10 degrees on the linkage map. Using mutants with alterations in large subunit ribosomal proteins L20 or L24, I mapped the corresponding genes near leuA at approximately 240 degrees. The data were fully consistent with the fact that the genes for the two proteins were close together but not near any other ribosomal protein genes, as is also the case with the genes for the corresponding proteins of Escherichia coli.     

Genes coding for ribosomal proteins S15, L21, and L27 map near argG in Escherichia coli.

Mutants with alterations in the structural genes for ribosomal proteins S15, L21, and L27 were used in mapping the genes coding for these proteins. Results from P1kc-mediated transductions indicate that the genes for L21 (rplU) and L27 (rpmA) form a gene cluster and are located between argG and gltB at 68.1 min, whereas the gene for S15 (rpsO) is situated close to, but on the opposite side or, argG. The gene order in this region is concluded to be gltB-(rplU, rpmA)-argG-rpsO-mtr.     

A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori

Two cDNAs encoding novel Rel proteins were cloned from the silkworm, Bombyx mori. These cDNA clones (BmRelA and BmRelB) showed identical nucleotide sequences except for the 5'-region. BmRelB cDNA derived probably from an alternatively spliced mRNA lacked 241 bp nucleotides at the 5'-region of the BmRelA cDNA, resulting in a loss of the first 52 amino acids. Expression of antibacterial peptide genes was strongly inhibited upon infection with Micrococcus luteus in transgenic silkworms in which BmRel gene expression was knocked down, suggesting that these two Rel proteins are involved in activation of antibacterial peptide genes. Co-transfection experiments indicated that BmRelB activated the Attacin gene strongly and other genes to a lesser extent, whereas BmRelA activated Lebocin 4 gene strongly and Attacin and Lebocin 3 genes very weakly. The Rel homology domain of BmRelA and BmRelB was shown to bind specifically to kappaB sites of antibacterial peptide genes. Proline-rich domains of the BmRels were necessary for activation of antibacterial peptide genes. These results illustrate that a minor structural change in Rel proteins can provoke a dramatic differential activation of antibacterial peptide genes, suggesting a novel regulatory mechanism for insect antibacterial peptide gene expression.     

Rules of nonallelic noncomplementation at the synapse in Caenorhabditis elegans

Nonallelic noncomplementation occurs when recessive mutations in two different loci fail to complement one another, in other words, the double heterozygote exhibits a phenotype. We observed that mutations in the genes encoding the physically interacting synaptic proteins UNC-13 and syntaxin/UNC-64 failed to complement one another in the nematode Caenorhabditis elegans. Noncomplementation was not observed between null alleles of these genes and thus this genetic interaction does not occur with a simple decrease in dosage at the two loci. However, noncomplementation was observed if at least one gene encoded a partially functional gene product. Thus, this genetic interaction requires a poisonous gene product to sensitize the genetic background. Nonallelic noncomplementation was not limited to interacting proteins: Although the strongest effects were observed between loci encoding gene products that bind to one another, interactions were also observed between proteins that do not directly interact but are members of the same complex. We also observed noncomplementation between genes that function at distant points in the same pathway, implying that physical interactions are not required for nonallelic noncomplementation. Finally, we observed that mutations in genes that function in different processes such as neurotransmitter synthesis or synaptic development complement one another. Thus, this genetic interaction is specific for genes acting in the same pathway, that is, for genes acting in synaptic vesicle trafficking.     

A possible origin of newly-born bacterial genes: significance of GC-rich nonstop frame on antisense strand.

Base compositions were examined at every position in codons of more than 50 genes from taxonomically different bacteria and of the corresponding antisense sequences on the bacterial genes. We propose that the nonstop frame on antisense strand [NSF(a)] of GC-rich bacterial genes is the most promising sequence for newly-born genes. Reasons are: (i) NSF(a) frequently appears on the antisense strand of GC-rich bacterial genes; (ii) base compositions at three positions in the codon are nearly symmetrical between the gene having around 55% GC content and the corresponding NSF(a); (iii) amino acid compositions of actual proteins are also similar to those of hypothetical proteins from the GC-rich NSF(a); and (iv) proteins from NSF(a) of 60% or more GC content are flexible enough to adapt to various molecules encountered as novel substrates, due to the high glycine content. To support our proposition, using a computer we generated hypothetical antisense sequences with the same base compositions as of NSF(a) at each base position in the codon, and examined properties of resulting proteins encoded by the imaginary genes. It was confirmed that NSF(a) of GC-rich gene carrying about 60% GC content is competent enough for a newly-born gene.     

Overlapping functions of components of a bacterial Sec-independent protein export pathway.

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.     

Preparation of recombinant proteins in milk to improve human and animal health

Milk is a very abundant source of proteins for animal and human consumption. Milk composition can be modified using transgenesis, including exogenous gene addition and endogenous gene inactivation. The study of milk protein genes has provided researchers with regulatory regions capable of efficiently and specifically driving the expression of foreign genes in milk. The projects underway are aimed at modifying milk composition, improving its nutritional value, reducing mammary infections, providing consumers with antipathogen proteins and preparing purified recombinant proteins for pharmaceutical use. The present paper summarises the current progress in this field.     

Evolution of antiparallel two-domain membrane proteins: tracing multiple gene duplication events in the DUF606 family

X-ray crystallography has revealed that many integral membrane proteins consist of two domains with a similar fold but opposite (antiparallel) orientation in the membrane. The proteins are believed to have evolved by gene duplication and gene fusion events from a dual topology ancestral membrane protein, that adapted both orientations in the membrane and formed antiparallel homodimers. Here, we present a detailed analysis of the DUF606 family of bacterial membrane proteins that contains the entire collection of intermediate states of such an evolutionary pathway: single genes that would code for dual topology homodimeric proteins, paired genes coding for homologous proteins with a fixed but opposite orientation in the membrane that would form heterodimers, and fused genes that encode antiparallel two-domain fusion proteins. Two types of paired genes can be discriminated corresponding to the order in which the genes coding for the two oppositely oriented proteins occur in the operon. On the protein level, the heterodimers resulting from the two types of gene pairs are indistinguishable. In contrast, two types of fused genes corresponding to the two possible orders in which the oppositely oriented domains are present in the encoded proteins, do result in discernible types of proteins. The large number of genetic and protein states in the DUF606 family allowed for a detailed phylogenic analysis that revealed a total of nine independent duplication events in the DUF606 family, five of which resulted in paired genes, and four resulted in fused genes. Noticeably, there was no evidence for a sequential mechanism in which fusions evolve from a pair of genes. Rather, an evolutionary mechanism is proposed by which antiparallel two-domain proteins are the direct result of a gene duplication event. Combining the phylogeny of proteins and hosting microorganisms allowed for a reconstruction of the evolutionary pathway.     

Measurement of gene number for seed storage proteins in Pisum.

We have measured the numbers of genes coding for the three seed storage proteins, vicilin, convicilin and legumin, in a number of Pisum genotypes of variant protein composition. No difference in gene number existed among P. sativum genotypes for any of the proteins. There were differences in the number of genes coding for individual proteins with approximately 11 genes (per haploid genome) for vicilin, 8 genes for legumin and 1 gene for convicilin.     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

Evolution of the primate lentiviruses: evidence from vpx and vpr.

The genomes of the four primate lentiviral groups are complex and contain several regulatory or accessory genes. Two of these genes, vpr and vpx, are found in various combinations within the four groups and encode proteins whose functions have yet to be elucidated. Comparison of the encoded protein sequences suggests that the vpx gene within the HIV-2 group arose by the duplication of an ancestral vpr gene within this group. Evolutionary distance analysis showed that both genes were well conserved when compared with viral regulatory genes, and indicated that the duplication occurred at approximately the same time as the HIV-2 group and the other primate lentivirus groups diverged from a common ancestor. Furthermore, although the SIVagm vpx proteins are homologous to the HIV-2 group vpx proteins, there are insufficient grounds from sequence analysis for classifying them as vpx proteins. Because of their similarity to the vpr proteins of other groups, we suggest reclassifying the SIVagm vpx gene as a vpr gene. This creates a simpler and more uniform picture of the genomic organization of the primate lentiviruses and allows the genomic organization of their common precursor to be defined; it probably contained five accessory genes: tat, rev, vif, nef and vpr.     

Determination of the chromosomal locations of four Bacillus subtilis genes which code for a family of small, acid-soluble spore proteins.

The chromosomal locations of four genes which code for small, acid-soluble spore proteins (SASP) in Bacillus subtilis have been determined. Although these four genes code for extremely homologous small, acid-soluble spore proteins (greater than 65% sequence identity), the genes are not clustered but are located at 70 degrees (adjacent to glyB [sspB gene]), 115 degrees (between metC and pyr cluster [sspD gene]), 180 degrees (between metB and kauA [sspC gene]), and 260 degrees (between ilvC and aroG [sspA gene]) on the B. subtilis genetic map.     

Telomeric associated sequences of Drosophila recruit polycomb-group proteins in vivo and can induce pairing-sensitive repression

In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. Here, we present the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes revealed a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE).     

The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.     

Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group.

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) was found to induce the synthesis of mRNA encoding a basic protein with a 67% amino acid sequence homology to the known acidic pathogenesis-related (PR) proteins 1a, 1b and 1c. By Southern blot hybridization it was shown that the tobacco genome contains at least eight genes for acidic PR-1 proteins and a similar number of genes encoding the basic homologues. Clones corresponding to three of the genes for acidic PR-1 proteins were isolated from a genomic library of Samsun NN tobacco. The nucleotide sequence of these genes and their flanking sequences were determined. One clone was found to correspond to the PR-1a gene; the two other clones do not correspond to known TMV-induced PR-1 mRNA's and may represent silent genes. Compared to the PR-1a gene, these genes contain an insertion or deletion in the putative promoter region and mutations affecting the PR-1 reading frame.