Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network

The sequence of paired, a pair-rule gene required for segmentation in Drosophila, is presented. A search for genes with domains homologous to the paired gene was initiated and three homologues from a set of 12 were characterized with respect to temporal or spatial expression and sequence homologies. All four are transcribed in early development, one in the oocyte and during cleavage stages in the form of a gradient. In addition to the prd-specific his-pro repeat, some of the 12 genes contain M-repeats and two new types of homeo boxes not detectable by hybridization with the two known classes of homeo boxes. The observed linking of gene sets through combinations of homologies coding for protein domains is consistent with a general network concept of gene action.     

Towards a collective biology of the gene

Two features of heterochromatin: heteropycnosis (high values of chromatin condensation), and repressed genetic expression, force upon us the use of cooperative variables, rather than molecular ones. In particular a "repressor" hypothesis is formulated, in which a useful parameter is clearly identified. This enables us to discuss the synchronized repression of a large number, n, of genes (as in the case of the Barr body, in which n is larger than 100). The hypothesis is documented with phenomena known to occur in active chromatin. Possible tests are suggested.     

Determining the evolutionary potential of a gene

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base- substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta- galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.      

The we gene is a modifier of the wal gene in mice

Interaction of gene wellhaarig (we) with genes waved alopecia (wal) and hairless (hr) was studied in mice. The mutant gene we is responsible for the development of a specific waved coat in homozygotes. Homozygous mice carrying mutant gene wal also have a wavy coat, though a partial alopecia develops with time in these animals. In homozygotes for the hr gene, hair loss is observed beginning from the age of ten days. A series of crosses we/we and wal/wal yielded animals with we/+wal/wal and we/we wal/wal genotypes. In mice we/+wal/wal carrying gene we at a single dose, alopecia is accelerated significantly as compared to the single-dose homozygotes +/+wal/wal. In we/we wal/wal mice, alopecia starts earlier than in we/+wal/wal mice; by the age of one month, the double homozygotes are almost hairless except for small body areas covered with a sparse coat. In addition, curliness of the first-generation hair in mice we/we wal/wal is much more expressed than in +/+wal/wal and we/we+/+ mice. The obtained evidence suggests that the we gene is a modifier of the wal gene because the former enhances the effects of the wal gene, which is confirmed by the earlier onset of alopecia and progression of the latter in mice having the we/+wal/wal genotype and especially in we/we wal/wal animals. The we/we hr/+ mice do not differ in coat from we/we+/+ mice; in both cases, the coat is wavy. The coat of double homozygotes we/we hr/hr, is similar to that of we/we+/+ mice until ten days of age, when the signs of alopecia appear. By the age of 21 days, mice we/we hr/hr have lost their coat completely like mice +/+ hr/hr. Hence, the we gene is a modifier of the wal gene though it does not interact with hr gene during the coat formation.     

The use of the luxA gene of the bacterial luciferase operon as a reporter gene

Summary Bacterial luciferase can be assayed rapidly and with high sensitivity both in vivo and in vitro. Here we demonstrate that the N-terminal hydrophobic domain of the catalytic subunit of the luciferase enzyme is indispensable for enzyme activity, although N-terminal translational fusions with full luciferase activity can be obtained. Bacterial luciferase is therefore ideally suited as a reporter enzyme for gene fusion experiments. A list of vectors for the convenient use of the luciferase marker genes to monitor gene expression in vivo are presented.     

Contribution of lateral gene transfer to the gene repertoire of a gut-adapted methanogen

Methanobrevibacter smithii is the most abundant archaeon in the human colon. As most of its neighbors are bacterial species, it is expected that lateral gene acquisition from bacteria might have contributed to the evolution and adaptation of this archaeon. We performed a tree-based genome-wide survey of putative lateral gene transfer products in M. smithii, using a phylogenetic pipeline. Over 15% of the coding genes of M. smithii are inferred to be bacterial in origin, based on this analysis. Laterally acquired genes have had the largest contribution to surface functions, and encode glycosyl-transferases and adhesin-like proteins. In addition, several important ABC transporters, especially metal transporters are of bacterial origin. Thus, bacterial genes contributed to the host-adaptation by allowing a larger variety of surface structures and increasing the efficiency of metal ion uptake in the competitive gut niche.     

The sequence of a sea urchin muscle actin gene suggests a gene conversion with a cytoskeletal actin gene

Summary We report the nucleotide sequence of the single muscle actin gene of the sea urchinStrongylocentrotus purpuratus. Comparison of the protein-coding sequence of this muscle actin gene (pSpG28) with that of two linked sea urchin cytoskeletal actin genes (pSpG17 and CyIIa) reveals a region of exceptional sequence conservation from codon 61 through codon 120. Furthermore, when silent nucleotide changes are compared, the conservation of this region is still evident (7.9% silent site differences in the conserved region vs 43.3% silent site differences in the rest of the gene when pSpG28 and CyIIa are compared), indicating that the conservation is not due to particularly stringent selection on the portion of the protein encoded by this region of the genes. These observations suggest that a gene conversion has occurred between the muscle actin gene and a cytoskeletal actin gene recently in the evolution of the sea urchin genome. Gene conversion between nonallelic actin genes may thus play a role in maintaining the homogeneity of this highly conserved gene family.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus. This revised version was published online in July 2006 with corrections to the Cover Date.     

A dual origin of the Xist gene from a protein-coding gene and a set of transposable elements

X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding genes. In this study we show that the key XIC gene Xist, which displays fragmentary homology to a protein-coding gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The Xist gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 gene. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into Xist accompanies the overall evolution of the gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the Xist gene and is found in other XIC genes producing non-coding nuclear RNA.     

Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

Recombination of the GFP gene to the BFP gene using a man-made site-selective DNA cutter

By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196–198, TAT at 199–201, and ACC at 502–504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.