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.     

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.     

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.     

Chromosomal localization of the human apoprotein CI gene and of a polymorphic apoprotein AII gene

Human apoprotein(apo) CI and apo AII cDNA probes have been used to analyze the segregation of the human genes in panels of human-mouse hybrids. The apo CI (APOCI) gene segregates with chromosome 19 and the apo AII (APOA2) gene with chromosome 1. Somatic cell hybrids containing chromosome translocations were used to map the apo AII gene to the 1p21-1qter region. Human APOA2 is polymorphic for the restriction endonuclease Msp I. Comparison of human and mouse chromosome 1 reveals a conserved group including apo AII, renin and peptidase genes and suggests that APOA2 will be found distal to this group on human chromosome 1. The mouse apo AII gene is closely linked with genes that regulate HDL structure. Similar HDL regulatory genes will probably be found near human APOA2.     

A HindIII RFLP and a gene lesion in the coagulation factor VIII gene

Summary The presence and inheritance of restriction fragment length polymorphisms (RFLPs) and gene lesions in the coagulation factor VIII gene were investigated in 15 hemophilia families. An abnormal HindIII 2.6-kb band, previously detected in a severe hemophiliac, was observed in a not severely affected patient and also in the normal gene of a woman carrying a hemophilic gene in which the lesions was found. The TaqI site in exon 24 of this defective gene was removed by a C to T transition causing an amino acid change (ArgGln). Very low amounts of factor VIII activity and antigen were detected in the severely affected grandson. The presence of the HindIII 2.6-kb fragment in both normal and patholgoical genes indicates that a factor VIII RFLP without functional meaning was found. Its frequency, determined in 60 chromosomes, is 0.18. Double digestions enabled us to map the polymorphic site 3 to the exon 19.     

Analysis of the Salmonella fim gene cluster: Identification of a new gene (fimI) encoding a fimbrin-like protein and located downstream from the fimA gene

Abstract The fimA gene coding for the major component (fimbrin) of type 1 fimbriae was mapped within the Salmonella typhi fim gene cluster, and its nucleotide sequence determined. The deduced amino acid sequence of S. typhi fimbrin is highly homologous to that of S. typhimurium type 1 fimbrin and showed similarity to that of other enterobacterial type 1 fimbrins. Downstream of fimA , an open reading frame was found, named fimI , able to encode a fimbrin-like protein. The fimI product could represent the counterpart, in type 1 fimbriae, of the PapH protein involved in cell anchoring and length modulation of Escherichia coli Pap pili. This genetic organization was found to be common to other Salmonella serovars, including S. typhimurium and S. choleraesuis .     

Cloning and expression analysis of a MAPKKK gene and a novel nodulin gene of Lotus japonicus

We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.     

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.     

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects.The 1.9 kb 5-upstream fragment (–1559 to +342) of the CatA gene was fused with the Escherichia coli -glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between –1564 to –699. Abscisic acid (ABA) at a final concentration of 10^–6 M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at –266 to –254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light.The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM 110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.author for corresondenceThe nucleotide sequence data reported will appear in the DDBJ EMBL and GenBank Nucleotide Sequence Databases under the accession number D29966.     

Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product

A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.     

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.      

Isolation and molecular characterization of rasfadin, a novel gene in the vicinity of the bovine prion gene

A novel gene, rasfadin (RASSF2) was identified close to the bovine prion gene, and its genomic structure was derived with a combination of exon trapping and RACE. The gene covers at least 28 kb and maps to the same chromosomal region as the prion gene in cattle, sheep, and human. The RASSF2 ORF is composed of 987 base pairs divided into nine exons and shows a high nucleotide (88%) and amino acid similarity (95%) with a previously described human cDNA, KIAA0168. The bovine 3′UTR region is significantly shorter than the human counterpart, but shares with it two highly conserved nucleotide blocks. The expression of the gene was investigated in brain, liver, and spleen. Alternative splicing yields a shorter product in the liver composed of only four exons. Computer analysis showed a highly significant similarity of the rasfadin protein with the Ras association (RalGDS/AF-6) domain family 2 and with the afadin family, respectively, for the longer brain/spleen and the shorter liver variants. Received: 26 June 2000 / Accepted: 8 September 2000     

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.