Epistatic interactions between loci of one-carbon metabolism modulate susceptibility to breast cancer

In view of growing body of evidence substantiating the role of aberrations in one-carbon metabolism in the pathophysiology of breast cancer and lack of studies on gene–gene interactions, we investigated the role of dietary micronutrients and eight functional polymorphisms of one-carbon metabolism in modulating the breast cancer risk in 244 case–control pairs of Indian women and explored possible gene–gene interactions using Multifactor dimensionality reduction analysis (MDR). Dietary micronutrient status was assessed using the validated Food Frequency Questionnaire. Genotyping was done for glutamate carboxypeptidase II (GCPII) C1561T, reduced folate carrier (RFC)1 G80A, cytosolic serine hydroxymethyltransferase (cSHMT) C1420T, thymidylate synthase (TYMS) 5′-UTR tandem repeat, TYMS 3′-UTR ins6/del6, methylenetetrahydrofolate reductase (MTHFR) C677T, methyltetrahydrofolate-homocysteine methyltransferase (MTR) A2756G, methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) A66G polymorphisms by using the PCR-RFLP/AFLP methods. Low dietary folate intake (P < 0.001), RFC1 G80A (OR: 1.38, 95% CI 1.06–1.81) and MTHFR C677T (OR: 1.74 (1.11–2.73) were independently associated with the breast cancer risk whereas cSHMT C1420T conferred protection (OR: 0.72, 95% CI 0.55–0.94). MDR analysis demonstrated a significant tri-variate interaction among RFC1 80, MTHFR 677 and TYMS 5′-UTR loci (P _trend < 0.02) with high-risk genotype combination showing inflated risk for breast cancer (OR 4.65, 95% CI 1.77–12.24). To conclude, dietary as well as genetic factors were found to influence susceptibility to breast cancer. Further, the current study highlighted the importance of multi-loci analyses over the single-locus analysis towards establishing the epistatic interactions between loci of one-carbon metabolism modulate susceptibility to the breast cancer.     

Stress proteins of<Emphasis Type="Italic"> Clostridium perfringens</Emphasis> type A immunoreact with antiserum from rabbits infected with gas gangrene

Various stressors were used to induce stress proteins in Clostridium perfringens. Cultures of C. perfringens FD-1041 were subjected to cold shock (28°C for 1 h), acid shock (pH 4.5 for 30 min), or heat shock (50°C for 30 min). Cells were lysed and protein samples were analyzed by immunoblotting with antiserum derived from rabbits suffering from gas gangrene. Eight cold shock proteins (approximate M_r 101, 82, 70, 37, 22, 12, 10 and 6 kDa) and also eight heat shock proteins (approximate M_r 101, 82, 70, 27, 22, 16, 12 and 10 kDa) were immunoreactive with the serum. No immunoreactive proteins were detected in samples subjected to acid shock proteins and purified DnaK protein was also non-immunoreactive with the serum. These immunogenic stress proteins may be important in regulating diseases caused by C. perfringens. Such proteins could be involved in cell survival mechanisms, serve as targets during infection, or play a role in recognition of the bacteria by the host.     

Lack of an HSP70 heat shock response in two Antarctic marine invertebrates

Members of the HSP70 gene family comprising the inducible (HSP70) genes and GRP78 (glucose-regulated protein 78 kDa) were identified in an Antarctic sea star (Odontaster validus) and an Antarctic gammarid (Paraceradocus gibber). These genes were surveyed for expression levels via Q-PCR after an acute 2-hour heat shock experiment in both animals and a time course assay in O. validus. No significant up-regulation was detected for any of the genes in either of the animals during the acute heat shock. The time course experiment in O. validus produced slightly different results with an initial down regulation in these genes at 2°C, but no significant up-regulation of the genes either at 2 or 6°C. Therefore, the classical heat shock response is absent in both species. The data is discussed in the context of the organisms’ thermal tolerance and the applicability of HSP70 to monitor thermal stress in Antarctic marine organisms.     

Linkage analysis of HSP70 genes and historecognition locus in Botryllus schlosseri

 The protochordate allorecognition system has long invited comparison with the vertebrate major histocompatibility complex (MHC). In the colonial species Botryllus schlosseri, a rapid fusion or rejection response resembling graft acceptance or rejection in vertebrates is controlled by a single highly polymorphic genetic region. Because linkage between heat shock protein 70 (HSP70) genes and the MHC appears to be conserved within the vertebrate lineage, linkage relationships between two HSP70 genes (HSP70.1 and HSP70.2) and the historecognition locus (FuHC) have been analyzed in B. schlosseri. Segregation patterns of restriction fragment length polymorphisms located in the 3′ flanking regions of HSP70.1 and HSP70.2 were determined for progeny of defined crosses. These progeny were also analyzed for fusibility type by an in vivo cut colony assay. No close linkage was detected between any of the three loci. These results do not support the hypothesis that the allorecognition response in B. schlosseri is determined by an MHC homologue. However, it remains a possibility that orthologues of other MHC-linked genes will be linked to the B. schlosseri FuHC. Received: 29 June 1997 / Revised: 6 October 1997     

Seasonal variations in biomass, abundance and composition of zooplankton in the subarctic waters north of Iceland

The seasonal variations in biomass, abundance and species composition of zooplankton in relation to hydrography and chlorophyll a were studied in the subarctic waters north of Iceland. The sampling was carried out at approximately monthly intervals from February 1993 to February 1994 at eight stations arranged along a transect extending from 66°16′N–18°50′W to 68°00′N–18°50′W. The mean temperature at 50 m depth showed a clear seasonal pattern, with lowest water temperatures in February (∼1.1°C) and the highest in July (∼5.4°C). The spring growth of the phytoplankton began in late March and culminated during mid-April (∼7.0 mg Chl a m⁻³). Both the biomass and the abundance of total zooplankton were low during the winter and peaked once during the summer in late May (∼4 g m⁻² and ∼38,000 individuals m⁻²). A total of 42 species and taxonomic groups were identified in the samples. Eight taxa contributed ∼90% of the total zooplankton number. Of these Calanus finmarchicus was by far the most abundant species (∼60% of the total zooplankton). Less important groups were ophiuroid larvae (∼9%), Pseudocalanus spp. (∼8%), Metridia longa (∼4%), C. hyperboreus (∼3%), Acartia longiremis (∼2%), chaetognaths (∼2%) and euphausiid larvae (∼2%). The dominant copepods showed two main patterns in seasonal abundance: C. finmarchicus, C. hyperboreus and C. glacialis had one annual peak in numbers in late May, while Pseudocalanus spp., M. longa and A. longiremis showed two maxima during the summer (July) and autumn (October/November). Ophiuroid larvae and chaetognaths (mainly Sagitta elegans) peaked during the middle of July, while the number of euphausiid eggs and larvae was greatest from May to July. The succession in population structure of C. finmarchicus indicated its main spawning to be in April and May, coincident with the phytoplankton spring bloom. A minor spawning was also observed sometime between August and October. However, the offspring from this second spawning contributed only insignificantly to the overwintering stock of C. finmarchicus. Received: 12 September 1997 / Accepted: 1 March 1998     

PCR Genome Walking Identifies a Genetic Locus Comprising Two Heat Shock Genes (hslV and hslU) from Leptospira borgpetersenii Serovar Hardjobovis

PCR walking on genomic DNA of Leptospira borgpetersenii serovar hardjobovis has identified a genetic locus comprising two heat shock genes (hslV and hslU). This is the first molecular study on hslV and hslU in a Leptospira species. The hslV gene has an open reading frame (ORF) of 543 bp coding for a 180-amino acid polypeptide (19.476 kD) with 49–62% identity to other bacterial HslV homologs. Forty-three nucleotides downstream from the hslV stop codon was found a second heat shock gene hslU with an ORF of 1401 bp encoding a 53.139-kD polypeptide of 467 amino acids with 49–56% identity to the HslU homologs from other bacteria. An Escherichia coli-like ribosome binding site (RBS) and a ς³²-like heat shock promoter sequence were present upstream of both heat shock genes. Identification of the hslV and hslU genes in a Leptospira species and the high degree of similarity of the encoded proteins to the E. coli homologs suggest that the encoded HslV and HslU proteins function as forming an ATP-dependent protease complex as in E. coli. Received: 7 March 2001 / Accepted: 3 May 2001     

Genome‐wide identification and characterization of HSP gene superfamily in whitefly (Bemisia tabaci) and expression profiling analysis under temperature stress

Heat shock proteins (HSP)are essential molecular chaperones that play important roles in the stress stimulation of insects.Bemisia tabaci,a phloem feeder and invasive species,can cause extensive crop damage through direct feeding and transmission of plant viruses.Here we employed comprehensive genomics approaches to identity HSP superfamily members in the Middle East Asia Minor 1 whitefly genome.In total,we identified 26 Hsp genes,including three Hsp90,17 Hsp70,one Hsp60 and five sHSP (small heat shock protein)genes.The HSP gene superfamily of whitefly is expanded compared with the other five insects surveyed here.The gene structures among the same families are relatively conserved.Meanwhile,the motif compositions and secondary structures of BtHsp proteins were predicted.In addition,quantitative polymerase chain reaction analysis showed that the expression patterns of BtHsp gene superfamily were diverse across different tissues of whiteflies.Most Hsp genes were induced or repressed by thermal stress (40℃)and cold treatment (4℃)in whitefly.Silencing the expression of BtHsp70-6 significantly decreased the survival rate of whitefly under 45℃.All the results showed the Hsps conferred thermo-tolerance or cold-tolerance to whiteflies that protect them from being affected by detrimental temperature conditions.Our observations highlighted the molecular evolutionary properties and the response mechanism to temperature assaults of Hsp genes in whitefly.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (σ ³²) factors from other bacterial species. It was not possible to inactivate the R. capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures. Received: 16 March 1998 / Accepted: 28 July 1998     

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L

A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length cDNA of CiHsp70 was 2,232 bp, consisting of a 5′-terminal untranslated region (UTR) of 76 bp, a 3′-terminal UTR of 203 bp with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17 amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or 93‰ NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment.     

Regulatory Sequences of Orthologous petD Chloroplast mRNAs are Highly Specific among Chlamydomonas Species

The 5′ untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5′ UTR. To further analyze these elements, we compared 5′ UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5′ UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5′ UTRs was tested in C. reinhardtii chloroplasts using β-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.     

Molecular diversity and catalytic activity of Thermus DNA polymerases

Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s. We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22 representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5′–3′ exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction.