Half Of Gene Research Articles

Phylogenetic Analysis of Ridgewayia (Copepoda: Calanoida) from the Galapagos and of a New Species from the Florida Keys With a Reevaluation of the Phylogeny of Calanoida

The mitochondrial gene cytochrome-c oxidase subunit 1 (COI) and the nuclear ribosomal DNA region known as Internal Transcribed Spacer 1 (ITS1) are used in a phylogenetic analysis of Ridgewayia from the Galapagos Islands and of a new species, Ridgewayia tortuga, from the Florida Keys. In addition, the phylogeny of Calanoida is reconstructed based on the 18S ribosomal RNA gene. The following characters exclude R. tortuga from the three recognized species groups of Ridgewayia: the presence of only 7 setae on the terminal endopod segment of leg 2; a 20-segmented male right antennule with two geniculations, the first between segments 9 and 10 and the second between segments 16 and 17; and details of the male fifth leg, in particular the elongate, unarmed, right endopod with a bifurcated tip. The molecular analysis shows that the first half of the COI gene not only fails to differentiate the various species of Ridgewayia, but it also fails to differentiate between the families Ridgewayiidae and Pseudocyclopidae. The second half of this gene and the ITS1 region are species specific. Molecular and morphological evidence suggest that the Galapagos ridgewayiids are the result of one colonization event and that the current phylogeography of these animals can be explained by a combination of vicariance and active migration models. The 18S ribosomal RNA gene proves successful in the reconstruction of the phylogeny of Calanoida with the following main conclusions: 1) Centropagoidea is the sister branch to all other Calanoida; 2) Ridgewayiidae and Pseudocyclopidae likely share a common ancestor with Augaptiloidea; 3) Ridgewayiidae and Pseudocyclopidae should be included in the same superfamily, the Pseudocyclopoidea; and 4) Spinocalanoidea likely needs to be included in Clausocalanoidea to recover the monophyly of the latter superfamily.

Huntingtin gene evolution in Chordata and its peculiar features in the ascidian Ciona genus

BackgroundTo gain insight into the evolutionary features of the huntingtin (htt) gene in Chordata, we have sequenced and characterized the full-length htt mRNA in the ascidian Ciona intestinalis, a basal chordate emerging as new invertebrate model organism. Moreover, taking advantage of the availability of genomic and EST sequences, the htt gene structure of a number of chordate species, including the cogeneric ascidian Ciona savignyi, and the vertebrates Xenopus and Gallus was reconstructed.ResultsThe C. intestinalis htt transcript exhibits some peculiar features, such as spliced leader trans-splicing in the 98 nt-long 5' untranslated region (UTR), an alternative splicing in the coding region, eight alternative polyadenylation sites, and no similarities of both 5' and 3'UTRs compared to homologs of the cogeneric C. savignyi. The predicted protein is 2946 amino acids long, shorter than its vertebrate homologs, and lacks the polyQ and the polyP stretches found in the the N-terminal regions of mammalian homologs. The exon-intron organization of the htt gene is almost identical among vertebrates, and significantly conserved between Ciona and vertebrates, allowing us to hypothesize an ancestral chordate gene consisting of at least 40 coding exons.ConclusionDuring chordate diversification, events of gain/loss, sliding, phase changes, and expansion of introns occurred in both vertebrate and ascidian lineages predominantly in the 5'-half of the htt gene, where there is also evidence of lineage-specific evolutionary dynamics in vertebrates. On the contrary, the 3'-half of the gene is highly conserved in all chordates at the level of both gene structure and protein sequence. Between the two Ciona species, a fast evolutionary rate and/or an early divergence time is suggested by the absence of significant similarity between UTRs, protein divergence comparable to that observed between mammals and fishes, and different distribution of repetitive elements.

Four Inteins and Three Group II Introns Encoded in a Bacterial Ribonucleotide Reductase Gene

A bacterial ribonucleotide reductase gene was found to encode four inteins and three group II introns in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The 13,650-bp ribonucleotide reductase gene is divided into eight extein- or exon-coding sequences that together encode a 768-amino acid mature ribonucleotide reductase protein, with 83% of the gene sequence encoding introns and inteins. The four inteins are encoded on the second half of the gene, and each has conserved sequence motifs for a protein-splicing domain and an endonuclease domain. These four inteins, together with known inteins, define five intein insertion sites in ribonucleotide reductase homologues. Two of the insertion sites are 10 amino acids apart and next to key catalytic residues of the enzyme. Protein-splicing activities of all four inteins were demonstrated in Escherichia coli. The four inteins coexist with three group II introns encoded on the first half of the same gene, which suggests a breakdown of the presumed barrier against intron insertion in this bacterial conserved protein-coding gene.

GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains.

Flowering of Arabidopsis is promoted by long days and delayed by short days. Mutations in the GIGANTEA (GI) gene delay flowering under long days but have little or no effect under short days. We have now isolated the GI gene and show that it encodes a novel, putative membrane protein. By comparing the sequence of the Arabidopsis gene with that of a likely rice orthologue and by sequencing mutant alleles, we identify regions of the GI protein that are likely to be important for its function. We show that GI expression is regulated by the circadian clock with a peak in transcript levels 8-10 h after dawn. The timing, height and duration of this peak are influenced by daylength. We analysed the interactions between GI and the LHY, CCA1 and ELF3 genes, previously shown to affect daylength responses; we show that the rhythmic pattern of GI expression is altered in the elf3, CCA1-OX and lhy genotypes, and that CCA1 and LHY expression are reduced by gi mutations. Our results are consistent with the idea that GI plays an important role in regulating the expression of flowering time genes during the promotion of flowering by photoperiod.

Reconstitution of an episomal mouse aprt gene as a consequence of recombination.

When a functional murine adenine phosphoribosyltransferase (aprt) gene linked to bovine papilloma virus (BPV) DNA is transfected into Aprt- L cells, the cells are rendered Aprt+ and the aprt gene persists as an episome. Cotransfection with two BPV vectors, one containing the 5' half of the aprt gene and the other the 3' half of the gene, that share about 300 bp of common sequence in intron 2, produces Aprt+ cells with functional aprt as an episome. Southern blot analysis of low molecular weight DNA derived from Hirt extracts revealed the regeneration of a diagnostic SmaI fragment, consistent with establishment of an episome with functional aprt that was reconstituted as a consequence of recombination. To establish cells with an episomal target for recombination, BPV vectors containing a G418 resistance marker and either the 5' half or 3' half of aprt were transfected into Aprt- L cells. Stably transfected cells, selected by their growth in G418, were in turn transfected with DNA containing the other half of the aprt gene. Following selection of Aprt+ cells, Southern blot and polymerase chain reaction (PCR) analysis of low molecular weight DNA confirmed the presence of a complete episomal aprt gene. The region of DNA shared by the episomal aprt fragment and the transfected aprt half was sequenced after PCR amplification of the reconstituted, episomal gene and was found to be wild type. The region of overlap that serves as the substrate for recombination lies entirely within an intron and can, therefore, tolerate nucleotide substitutions and deletions. The absence of such errors in the sequences examined is consistent with recombination events that are not error prone.

Structure of the murine lactotransferrin gene is similar to the structure of other transferrin-encoding genes and shares a putative regulatory region with the murine myeloperoxidase gene

The structure and nucleotide sequence of the murine lactotransferrin-encoding gene ( LTF) deduced partly by direct sequencing of genomic clones in the λ phage vector and partly by enzymatic amplification of genomic DNA segments primed with the oligodeoxyribonucleic primers homologous to the cDNA sequence. The λ phage clones contained the 5′ half of the gene corresponding to the first eight exons and an incomplete ninth exon interrupted by eight introns. Genomic clones corresponding to the 3′ half of the LTF gene could not be obtained on repeated attempts from two different mouse genomic libraries, suggesting the possible presence of unclonable sequences in this part of the gene. Hence, PCR was used to clone the rest of the gene. Four out of the presumed eight remaining introns were cloned along with the flanking exons using PCR. Comparison of the structure of the LTF gene with those of the two other known transferrin-encoding genes, human serum transferrin-encoding gene and chicken ovotransferrin-encoding gene reveals that all three genes have a very similar intron-exon distribution pattern. The hypothesis that the present-day transferrin-encoding genes have originated from duplication of a common ancestral gene is confirmed here at the gene level. An interesting finding is the identification of a region of shared nucleotides between the 5′ flanking regions of the murine LTF and myeloperoxidase-encoding genes, the two genes expressed specifically in neutrophilic granulocytes.

Genetic Analysis of H2, the Structural Gene for Phase-2 Flagellin in Salmonella

Non-flagellate H2 mutants were isolated from a phase-2 stable strain, SJW806 H1-gt- H2-enxon vh2-, a derivative of Salmonella typhimurium. By transductional crosses a deletion map and a recombination map of the H2 gene were made. There are three regions especially rich in nonflagellate mutational sites. By the use of the deletion map, mutational sites of 21 flagellar shape mutants were also determined. Most of them were located at two regions which coincide with two of the three regions rich in non-flagellate mutational sites. A gene, vh2, is closely linked to the promoter side of the H2 gene. Three-factor transductional crosses showed that the vh2 gene was on the left of the H2 gene in the present map. The H2 gene forms part of an operon with the distal gene rh1 which specifies the H1 repressor. Thus, a polarity effect of the H2 mutations on the expression of the rh1 gene was examined by observing whether a wild-type H1 allele introduced into the H2 mutants was expressed or not. Many of the H2 mutations were polar, and most of the strongly polar mutations were located in the left (promoter-proximal) half of the H2 gene, while most of the mutations in the right half of the gene were weakly polar or non-polar.

Deletion of the 3′ half of the yeast tRNA 3Leu gene does not abolish promoter function in vitro

The promoter function of the 3′ and 5′ half tRNA sequences in the yeast tRNA 3 Leu gene has been studied by in vitro transcription in Xenopus laevis germinal vesicle (GV) extracts. Truncation of the DNA template within the tRNA intervening sequence by Hpa I abolishes transcription. However, separation of the tRNA gene halves by insertion of a 300 bp DNA fragment at the Hpa I site does not affect the promoter efficiency. Further, the complete sequence of the 3′ half of the tRNA is not necessary for promoter function, because removal of the 3′ half of the gene by cleavage with Pvu II, within the DNA inserted at the Hpa I site, does not inhibit transcription.