Identification by gene deletion analysis of barS2, a gene involved in the biosynthesis of γ-butyrolactone autoregulator in Streptomyces virginiae

Virginiae butanolide (VB) is a member of the γ-butyrolactone autoregulators and triggers the production of streptogramin antibiotics virginiamycin M₁ and S in Streptomyces virginiae. A VB biosynthetic gene (barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound, and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW ₂ located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae. An erratum to this article can be found at     

Prion protein gene polymorphisms in sheep in the state of Paraná, Brazil

To determine the polymorphisms of the prion protein gene in sheep from the state of Paraná, Brazil, 323 animals of meat breeds (Suffolk, Hampshire Down, Texel, Ile de France, Dorper, Dorset, Santa Inês and crossbreds) were genotyped by restriction fragment length polymorphism (RFLP) analysis. The most frequent allele was ARQ, with a frequency of 0.61, followed by ARR (0.30). VRQ and AHQ alleles were present at very low frequencies (0.13 and 0.05 respectively), and the ARH allele was not found. Seven genotypes were identified (ARR/ARR, ARR/ARQ, ARQ/ARQ, ARR/VRQ, ARR/AHQ, ARQ/VRQ and ARQ/AHQ), of which ARQ/ARQ was the most frequent (0.41). The Santa Inês breed and crossbred animals showed the highest genotypic variability.     

Gene–environment and gene–gene interactions of specific MTHFR,MTR and CBS gene variants in relation to homocysteine in black South Africans

The methylenetetrahydrofolate reductase (MTHFR), cystathione-β-synthase (CBS) and methionine synthase (MTR) genes interact with each other and the environment. These interactions could influence homocysteine (Hcy) and diseases contingent thereon. We determined single nucleotide polymorphisms (SNPs) within these genes, their relationships and interactions with total Hcy concentrations within black South Africans to address the increased prevalence of diseases associated with Hcy. The MTHFR 677 TT and MTR 2756 AA genotypes were associated with higher Hcy concentrations (16.6 and 10.1 μmol/L; p < 0.05) compared to subjects harboring the MTHFR 677 CT/CC and the MTR 2756 AG genotypes (10.5, 9.7 and 9.5 μmol/L, respectively). The investigated CBS genotypes did not influence Hcy. We demonstrated interactions between the area of residence and the CBS T833C/844ins68 genotypes (p = 0.005) so that when harboring the wildtype allele, rural subjects had significantly higher Hcy than their urban counterparts, but when hosting the variant allele the environment made no difference to Hcy. Between the CBS T833C/844ins68 or G9276A and MTHFR C677T genotypes, there were two-way interactions (p = 0.003 and = 0.004, respectively), with regard to Hcy. Subjects harboring the MTHFR 677 TT genotype in combination with the CBS 833 TT/homozygous 844 non-insert or the MTHFR 677 TT genotype in combination with the CBS 9276 GA/GG displayed higher Hcy concentrations.     

Contribution of gene conversion in the evolution of the human β-like globin gene family

Gene conversion is referred to as one of two types of mechanisms known to act on gene families, mainly to maintain their sequence homogeneity or, in certain cases, to produce sequence diversity. The concept of gene conversion was established 20 years ago by researchers working with fungi. A few years later, gene conversion was also observed in the human genome, i.e. the γ-globin locus. The aim of this article is to emphasize the role of genetic recombination, particularly of gene conversion, in the evolution of the human β-like globin genes and further to summarize its contribution to the convergent evolution of the fetal globin genes. Finally, this article attempts to re-examine the origin and spread of specific mutations of the β-globin cluster, such as the sickle cell or β-thalassemia mutations, on the basis of repeated gene conversion events. Received: 13 February 1997 / Accepted: 15 May 1998     

Comparative gene mapping of lactoperoxidase,retinoblastoma, and α-lactalbumin genes in cattle,sheep, and goats

The lactoperoxidase (LPO), retinoblastoma (RB1), and -lactalbumin (LALBA) genes have been mapped by fluorescent in situ hybridization respectively to cattle Chromosomes (Chrs) 19, 12, 5; goat Chrs 19, 12, 5; and sheep Chrs 11, 10, 3. The results confirm the homologies among cattle, sheep, and goat chromosomes, previously reported, and provide more information for the comparison between the bovine and human karyotypes and gene maps.     

Characterization of β-casein gene in Indian riverine buffalo

The study aimed at characterization of buffalo β-casein gene and its promoter by PCR-SSCP analysis. Complete β-casein exon VII region analysis revealed two SSCP band patterns, with pattern-I representing predominant allele B (85%) present in homozygous (genotype BB) condition and pattern-II representing a rare allele A1 present in heterozygous condition (genotype A1B). Sequencing of two patterns revealed three nucleotide substitutions at codon 68, 151 and 193 of exon VII. The cDNA sequence of buffalo β-casein gene indicated three further nucleotide substitutions between allele A1 and B at codon 10, 39, and 41. Analysis of β-casein proximal promoter region (− 350 upstream to + 32) revealed four SSCP band patterns. These SSCP patterns corresponded to nucleotide substitutions at seven locations within 382 bp 5′ UTR region of β-casein gene. Haplotype analysis suggested pattern-I of exon VII (wild type) was associated with three types of promoters and pattern-II of exon VII (rare type) corresponded to one exclusive type of promoter. The study suggested two haplotypes of exon VII and four haplotypes of promoter for buffalo β-casein.     

Evaluation of seed storage-protein gene 5′ untranslated regions in enhancing gene expression in transgenic rice seed

5′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the β-glucuronidase (GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5′-UTRs of six seed storage-protein genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5′-UTRs significantly enhanced the expression level of the GluC promoter without altering its expression pattern. The 5′-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5′-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore, the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing the translational efficiencies of the mRNA. The 5′-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds.     

Molecular genetics of radiation-induced chromosome breaks in a gene area in Drosophila: "position" effect of gene mutation?

On the sample of 43 gamma-ray and neutron-induced inversion or translocation exchanges with the vestigial (vg) phenotype, the molecular cytogenetic analysis of distribution of exchange breakpoints on the molecular map of Drosophila vg region (subsection 49D3-4 on the polytene chromosome 2R) was performed using hybridisation in situ technique. Simultaneously, PCR-assay of DNA alterations in all exons and introns (except for intron 4) of the vg gene for 18 mutants with exchange breakpoints outside of the gene was carried out. The results obtained by these molecular genetic techniques have shown that 1) radiation-induced breaks under chromosome exchanges with the vg phenotype were regularly located inside of the vg gene (19 cases out of 43 studied ones or 44.2%) passing through the large introns; 2) breakpoints were frequently flanked by deletions of the gene as whole (3 exchanges) or of its major part (3 exchanges); 3) many of the breaks (18/43 or 41.8%) are situated outside (distal or proximal) of the gene although such mutants have got the vg phenotype; 4) 2/3 (12/18 or 66.7%) vg mutants with the breakpoint outside of gene show the intragenic DNA lesions (microdeletions, microinversions) occurring obviously independently and simultaneously with the neighbor chromosome breaks; 5) only each third vg mutant with break outside of the gene (6/18 or 33.3%) have the unchanged gene subregions under study and presents obviously the result of "position effect" which appear to manifest itself for a distance of 2-30 kb (more near and farther locations of the proximal and distal breakpoints, respectively, relative to the vg gene). Our findings showing regular induction of the multiple genetic lesions (chromosome breaks and mutations of the adjacent genes) on the both ends of chromosome exchange induced by single track produced by gamma-rays or neutrons were discussed as a scientific basis for the conceptually new approaches to the assessment of both genetic damage numbers in the cell genome with chromosome exchange (the multiple genetic lesions) and radiation genetic risk (our molecular genetic approach showing the need for an increase of risk levels at least on a factor of 3 for the heritable chromosome alterations detected by the ordinary cytogenetic monitoring).     

Mapping, expression and regulation of the TRα gene in porcine adipose tissue

Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRα and TRβ. The TRα isoform plays a critical role in mediating the action of thyroid hormone in adipose tissue. We mapped the porcine TRα gene to chromosome 12 p11-p13, by using the ImpRH panel. We examined tissue-localization of TRα and determined expression patterns of TRα in porcine adipose tissue with quantitative real-time PCR. TRα was expressed in all tissues, including heart, liver, spleen, stomach, pancreas, brain, small intestine, skeletal muscle, and subcutaneous adipose tissue. In the adipose tissue, the expression of TRα decreased postnatally. Compared to Yorkshire pigs, Jinhua pigs had significantly lower expression levels of TRα gene in the subcutaneous fat tissue. The expression levels of β2-AR, HSL and ATGL were also significantly lower in Jinhua pigs than in Yorkshire pigs. However, no significant differences in PPARγ and SREBP-1C expression levels were found between Jinhua and Yorkshire pigs. Incubation of porcine adipose tissue explants with high doses of isoproterenol (100 and 1000 nM) significantly increased the expression levels of TRα. We conclude that there is considerable evidence that TRα plays an important role in fat deposition in porcine adipose tissue.     

Effects of mutations in genesfadR,fabB, fadE,andenvC ofEscherichia coli on the action of the lysis gene of bacteriophageϕX174

The lytic effect of the expression of the cloned geneE of bacteriophage X174 inEscherichia coli is considerably amplified by a mutation in thefadR gene, which primarily affects the regulation of fatty acid degradation. In contrast, reduction of the fluidity of the cell membranes by use of thefabB andfadE mutations, which interfere with the synthesis and the oxidation of unsaturated fatty acids, severely inhibits the action of the X174 lysis gene product. A chain-forming mutant carrying a pleiotropic mutation in theenvC locus is also refractory to the X174 lysis protein. As shown by reversion and complementation of theenvC mutatation, a defect in at least one additional gene (rle) is involved in the generation of this refractoriness.     

Association mapping, patterns of linkage disequilibrium and selection in the vicinity of the PHYTOCHROME C gene in pearl millet

Key message

Linkage analysis confirmed the association in the region of PHYC in pearl millet. The comparison of genes found in this region suggests that PHYC is the best candidate.

Abstract

Major efforts are currently underway to dissect the phenotype–genotype relationship in plants and animals using existing populations. This method exploits historical recombinations accumulated in these populations. However, linkage disequilibrium sometimes extends over a relatively long distance, particularly in genomic regions containing polymorphisms that have been targets for selection. In this case, many genes in the region could be statistically associated with the trait shaped by the selected polymorphism. Statistical analyses could help in identifying the best candidate genes into such a region where an association is found. In a previous study, we proposed that a fragment of the PHYTOCHROME C gene (PHYC) is associated with flowering time and morphological variations in pearl millet. In the present study, we first performed linkage analyses using three pearl millet F₂ families to confirm the presence of a QTL in the vicinity of PHYC. We then analyzed a wider genomic region of ~100 kb around PHYC to pinpoint the gene that best explains the association with the trait in this region. A panel of 90 pearl millet inbred lines was used to assess the association. We used a Markov chain Monte Carlo approach to compare 75 markers distributed along this 100-kb region. We found the best candidate markers on the PHYC gene. Signatures of selection in this region were assessed in an independent data set and pointed to the same gene. These results foster confidence in the likely role of PHYC in phenotypic variation and encourage the development of functional studies.