Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Carotenoid synthesis in Streptomyces setonii ISP5395 is induced by the gene crtS, whose product is similar to a sigma factor

In many species of actinomycetes, carotenogenesis can be photoinduced. The capacity to respond to photoinduction is, however unstable and, in various strains of Streptomyces, is lost at a relatively high frequency. In Streptomyces setonii ISP5395, which normally produces no carotenoids, carotenoid-producing mutants can be obtained following protoplast regeneration. We report here the characterization of a gene, crtS, which was isolated from one such mutant and can confer on wild-type S. setonii ISP5395 cells the capacity to synthesize carotenoids. Sequence analysis of crtS reveals an open reading frame, which shows homology to genes that encode alternative sigma factors in Bacillus subtilis. We propose that crtS encodes a sigma factor which is necessary for the expression of a cryptic gene(s) for carotenoid biosynthesis in S. setonii ISP5395.     

Prediction of the halothane (Hal) genotypes of pigs by deducing Hal, Phi, Po2, Pgd haplotypes of parents and offspring: results from a large-scale practice in Swedish breeds

Results from a large-scale study, comprising 75 different breeding herds, are reported on predicting the halothane ( Hal ) genotypes of individual pigs by making use of the known close linkage between Hal and three C blood marker loci ( Phi, Po2, Pgd ). The parents haplotypes (involving Hal and marker loci) were determined from the HAL phenotypes (halothane test results) and marker loci phenotypes of their offspring in the first one or two litters studied. In subsequent litters of the Hal -marker loci haplotyped parents, the offspring's expected Hal genotypes could be predicted on the basis of the marker loci haplotypes inherited by them. By comparing the expected and observed HAL phenotypes of offspring in subsequent litters, the predicted Hal genotype was found to be correct in 90–95 % of the 4000 offspring (from Nn × Nn and Nn × nn matings) of Swedish Landrace and Yorkshire breeds studied.
The order of the three marker loci was confirmed as Phi-Po2-Pgd but the position of Hal with regards to Phi could not be resolved. The recombination frequencies between the most distant loci in this region, viz. Hal-Pgd and Phi-Pgd , were estimated to be 3–4.5 % and 4–6 % , respectively. The easy and rapid electrophoretic techniques described in the study to phenotype PHI, PO2, PGD, also allowed phe-notyping of six other polymorphic protein systems on the same gels. Thus Hal genotyping and effective parentage control can be conducted simultaneously.     

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5′-transposon end

1. Download : Download high-res image (250KB)
2. Download : Download full-size image

     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.     

Diversification of Hawaiian Cyrtandra (Gesneriaceae) under the influence of incomplete lineage sorting and hybridization

Cyrtandra (Gesneriaceae) is a genus of flowering plants with over 800 species distributed throughout Southeast Asia and the Pacific Islands. On the Hawaiian Islands, 60 named species and over 89 putative hybrids exist, most of which are identified on the basis of morphology. Despite many previous studies on the Hawaiian Cyrtandra lineage, questions regarding the reconciliation of morphology and genetics remain, many of which can be attributed to the relatively young age and evidence of hybridization between species. We utilized targeted enrichment, high‐throughput sequencing, and modern phylogenomics tools to test 31 Hawaiian Cyrtandra samples (22 species, two putative hybrids, four species with two samples each, one species with four samples) and two outgroups for species relationships and hybridization in the presence of incomplete lineage sorting (ILS). Both concatenated and species‐tree methods were used to reconstruct species relationships, and network analyses were conducted to test for hybridization. We expected to see high levels of ILS and putative hybrids intermediate to their parent species. Phylogenies reconstructed from the concatenated and species‐tree methods were highly incongruent, most likely due to high levels of incomplete lineage sorting. Network analyses inferred gene flow within this lineage, but not always between taxa that we expected. Multiple hybridizations were inferred, but many were on deeper branches of the island lineages suggesting a long history of hybridization. We demonstrated the utility of high‐throughput sequencing and a phylogenomic approach using 569 loci to understanding species relationships and gene flow in the presence of ILS.