Allelic polymorphism of mouse Igh-J locus,which encodes immunoglobulin heavy chain joining (JH) segments

The mouse genome contains four functional J _H genes, which encode immunoglobulin heavy chain joining segments. The J _H gene cluster is located a few kilobases 5 from the constant region genes (C genes) on chromosome 12. The polymerase chain reaction (PCR)-technique was used to amplify DNA stretches from mouse genome of approximately 1 340 nucleotides in length containing all four J _H genes (Igh-J locus). PCR products were directly used as templates in Sanger's dideoxy-sequencing, and sequences were determined. Twelve inbred mouse strains belonging to ten different Igh-C haplotypes were studied. The strains were: BALB/c, C58/J, RIII, DBA/2, CE, RF, CBA, NZB/J, AKR, C57BL/10, SJL, and A/J. Five allelic forms of the Igh-J locus were found among these strains. The A/J mouse has an allele (e) which differs from the BALB/c allele (a) by 15 nucleotides. C57BL and SJL have the allele (b) with eight differences from BALB/c. The CBA allele (j) has two differences, and the CE allele (f) has a single nucleotide difference compared with the BALB/c sequence. Based on the J _H , variable (V) and constant (C) region sequences we conclude that independent reshuffling of V _H ,J _H , and C _H gene clusters occurred during the evolution of Mus musculus.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X63146-X63175.     

甘薯肉桂酸-4-羟化酶基因克隆及序列分析

肉桂酸-4-羟化酶(Cinnamic acid-4-hydroxylase,C4H,EC 1.14.13.11)是苯丙烷途径中第二步反应酶,同时也是花色素苷前体生物合成途径中关键酶。该研究根据植物C4H的同源序列设计引物,通过RTPCR结合RACE的方法,在紫色甘薯中获得了与其相应的C4H基因,命名为Ib C4H(Gen Bank登录号GQ373157)。结果表明:(1)序列分析表明Ib C4H长1 668 bp,编码505个氨基酸,该氨基酸序列与其c DNA序列与Ib C4H蛋白与马铃薯C4H蛋白序列最为接近,与苹果、黑莓、大阿米芹、油菜一致性很高,均在70%以上。(2)二级结构预测表明α-螺旋和无规则卷曲是Ib C4H蛋白最大量的结构元件,而延伸链则散布于整个蛋白中。(3)三维结构建模预测,Ib C4H蛋白具备细胞色素P450氧和铁离子结合位点等典型的C4H结构。该研究结果为进一步了解花色素苷生物合成途径中的作用奠定了基础,也为花青素生物合成分子机理和代谢调控提供了靶位点和理论参考。     

Association of membranous nephropathy with T-cell receptor constant β chain and immunoglobulin heavy chain switch region polymorphisms

We have investigated T-cell antigen receptor constant chain genes (Tcr C ) and immunoglobulin (Ig) heavy chain switch region genes of HLA-DR-typed patients with membranous nephropathy (MN) employing DNA restriction fragment length polymorphism (RFLP) analysis. When a Tcr C probe in conjunction with the restriction endonuclease BgI II was used, a significant increase in the frequency of a 10.0;9.2 kb heterozygous RFLP phenotype was found in MN (75.0 % versus 42.1 in controls; P=0.002). When Sst I-restricted DNA from MN patients was hybridized with a DNA probe homologous to the switch region flanking the Ig C _µ heavy chain gene (S _µ), there was a significant decrease in the frequency of the 2.1; 2.6 kb heterozygous RFLP phenotype in MN (24.0% versus 54.6% in controls; P=0.004). These results suggest that Tcr beta and Ig heavy chain loci, as well as HLA antigens, may be important in the pathogenesis of MN.     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.     

A Biogeochemical and Genetic Survey of Acetylene Fermentation by Environmental Samples and Bacterial Isolates

Anoxic samples (sediment and groundwater) from 13 chemically diverse field sites were assayed for their ability to consume acetylene (C₂H₂). Over incubation periods ranging from ～ 10 to 80 days, selected samples from 7 of the 13 tested sites displayed significant C₂H₂ removal. No significant formation of ethylene was noted in these incubations; therefore, C₂H₂ consumption could be attributed to acetylene hydratase (AH) rather than nitrogenase activity. This putative AH (PAH) activity was observed in only 21% of the total of assayed samples, while amplification of AH genes from extracted DNA using degenerate primers derived from Pelobacter acetylenicus occurred in even fewer (9.8%) samples. Acetylene-fermenting bacteria were isolated as a pure culture from the sediments of a tidal mudflat in San Francisco Bay (SFB93) and as an enrichment culture from freshwater Searsville Lake (SV7). Comparison of 16S rDNA clone libraries revealed that SFB93 was closely related to P. carbolinicus, while SV7 consisted of several unrelated bacteria. AH gene was amplified from SFB93 but not SV7. The inability of the primers to generate amplicons in the SV7 enrichment, as well as from several of the environmental samples that displayed PAH activity, implied that either the primers were too highly constrained in their specificity or that there was a different type of AH gene in these environmental samples than occurs in P. acetylenicus. The significance of this work with regard to the search for life in the outer Solar System, where C₂H₂ is abundant, is discussed.     

Improved group‐specific primers based on the full SILVA 16S rRNA gene reference database

Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T‐RFLP) analysis, are well‐suited techniques for the examination of microbial community structures. The use of phylum‐ and class‐specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain‐specific primers. To date, several phylum‐ and class‐specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non‐target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T‐RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above‐mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics.     

Tetrahydrofolate-specific enzymes in <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> and growth dependence of this methanogenic archaeon on folic acid or <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H₄SPT) rather than tetrahydrofolate (H₄F) as a pterin C₁ carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H₄F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N⁵,N¹⁰-methylene-H₄F dehydrogenase/N⁵,N¹⁰-methenyl-H₄F cyclohydrolase) and GlyA (serine:H₄F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H₄F and H₄F, respectively (apparent K_m below 5 M). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H₄F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H₄F. From the p-aminobenzoic acid requirement, an intracellular H₄F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H₄SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H₄SPT and H₄F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C₁ pools in these methanogens.     

Non-allelic interaction conditioning spikelet sterility in an F2 population of indica/japonica cross in rice

Significant segregation of spikelet fertility occurred in an F₂ population derived from a spikelet fertility-normal F₁ hybrid produced by a cross between Palawan, a japonica variety, and IR42, an indica variety. To identify factors controlling the fertility segregation, we used 104 RFLP markers covering all 12 rice chromosomes to investigate the association of spikelet fertility and marker segregation. We found that the segregation of two sets of gene pairs was significantly (P < 0.001) associated with fertility segregation. The first pair of genes was linked to RFLP marker RG778 on chromosome 12 and RFLP markers RG690/RG369 on chromosome 1. A significant reduction in fertility was observed when the plants were homozygote at RG778 with the indica allele as well as homozygote at RG690/RG369 with the japonica allele. The second pair of genes was linked to RG218 on chromosome 12 and RG650 on chromosome 7, respectively. The recombinant homozygote at these two loci showed a significant reduction on spikelet fertility. The non-allelic interaction effect was further modified by a gene linked to RG778, resulting in even lower fertility. The results of this study provides the first evidence of chromosomal localization of sporophytic sterility genes whose interaction can result in a reduction of spikelet fertility in the F₂ derived from fertility-normal F₁.     

Methanogenic fermentation of benzoate in an enrichment culture

Enrichment cultures inoculated with black mud fermented benzoate according to the stoichiometric equation: 4 C₆H₅CO₂H+18 H₂O 15 CH₄+13 CO₂.Trans-2-hydroxycyclohexanecarboxylate, 2-oxo-cyclohexanecarboxylate, pimelate, caproate, butyrate, acetate, and molecular hydrogen were shown to be regular components of the culture fluid occurring in low concentrations. Inhibition of methanogenesis by chloroform, 4-chlorobutyrate, or 2-bromooctanoate resulted in a cessation of the benzoate breakdown after all intermediates had accumulated. It is proposed that benzoate is fermented via a direct reductive pathway to butyrate, acetate, H₂, and CO₂, whereafter butyrate is converted to acetate and H₂, and the latter substrates are fermented to CH₄ and CO₂ by methane producers.     

Generation of glyoxylate in methylotrophic bacteria

The mode of glyoxylate production from acetyl-CoA was investigated in three strains of methylotrophic bacteria,Pseudomonas MA,Pseudomonas AM1 and organism PAR. This investigation was prompted by the recently reported discovery of a homoisocitrate lyase in methylotrophic bacteria and the suggested involvement of this novel enzyme in assimilation of C₁ and C₂ compounds as part of a homoisocitrate-glyoxylate cycle. We were unable to detect cleavage of any of the four stereoisomers of homoisocitric acid by cell-free extracts of C₁-or C₂-grown bacteria. Extracts of C₁-grown bacteria did not catalyze condensation of glyoxylate with glutarate or production of glyoxylate from acetyl-CoA and 2-ketoglutarate. Extracts of C₁-grownPseudomonas MA catalyzed cleavage of isocitrate;threo-homoisocitrate was a potent competitive inhibitor of this reaction. These results indicate that homoisocitrate cleavage does not occur in any of the methylotrophs tested. The pathway for oxidation of acetyl-CoA to glyoxylate inPseudomonas AM1 and organism PAR therefore remains obscure.     

Effects of 2,5-norbornadiene on cocklebur seed germination and rice coleoptile elongation in response to CO2 and C2H4

To examine in more detail the mechanisms of cocklebur (Xanthium pennsylvanicum Wallr.) seed germination and rice (Oryza sativa L. cv. Sasanishiki) coleoptile elongation that were responsive to both C₂H₄ and CO₂, the effects of NBD (2,5-norbornadiene), a cyclic olefin known as a competitive inhibitor of C₂H₄, on those phenomena were tested under various conditions. NBD strongly inhibited germination of cocklebur seeds and their axial and cotyledonary growth. The NBD effects were significantly negated by endogenously evolved and exogenously applied CO₂ regardless of incubation temperature. Similarly, the inhibitory NBD effect was negated by C₂H₄ at 23°C, but at 33°C a low concentration (3 1/L) of C₂H₄ rather enhanced the inhibitory NBD effect. This phenomenon reflected the growth responses of the tip zone of axial tissues in cocklebur seeds to NBD and C₂H₄, in which both gases were antagonistic in regulating the axial growth at 23°C but additive in inhibiting it at 33°C. Maximal negation of these inhibitory NBD effects was brought about by simultaneous application of CO₂ and C₂H₄. Similarly, elongation of rice coleoptiles was suppressed by NBD, and when they were immature, its inhibitory action was counteracted by both C₂H₄ and CO₂, especially during simultaneous application. However, the inhibitory NBD effect was completely negated by C₂H₄ applied alone at concentrations above 500 1/L regardless of the physiological age of coleoptiles. These inhibitory NBD effects are additional evidence suggesting that C₂H₄ acts as a growth regulator in both cocklebur seed germination and rice coleoptile elongation. That NBD was capable of counteracting CO₂ action in some cases but was incapable of negating inhibitory C₂H₄ action, such as that observed in cocklebur seeds, suggests that NBD acts with some side effects besides being a competitive inhibitor of C₂H₄ actions.     

In vitro anti-leishmanial activities of germatranyl and Silicon incorporated diorganotin derivatives: Synthesis and spectroscopic properties

A series of germanium and silicon incorporated diorganotin derivatives of general formula where R¹ = H₃C, C₆H₅, p-CH₃C₆H₄, p-FC₆H₄; R² = H₂CSi(CH₃)₂C₆H₅, H₂CC₆H₅, p-CH₃C₇H₇ were synthesized by the reaction of appropriate diorganotin dichlorides and germatranyl (substituted) propionic acid in 1:2 mole ratio, respectively. The evidence regarding their structure is mainly based on spectroscopic data obtained by multinuclear (¹H, ¹³C, ²⁹Si, ¹¹⁹Sn) NMR and ^119mSn Mössbauer, IR and mass spectral studies in combination with melting points and elemental analyses. The compounds have been screened for in vitro anti-leishmanial activity against promastigotes of Leishmania major and the results offer potent activities which are better than the standard drug, pentamidine, for one compound.     

The Fractionation of Chlorine Isotopes by the Aerobic Methylotrophic Bacterium Methylobacterium dichloromethanicum Grown on Dichloromethane

Methylobacterium dichloromethanicum was found to be able to utilize dichloromethane (DCM) as the source of carbon and energy with the production of biomass, CO₂, and HCl. A comparative analysis of the abundances of the major DCM isotopomers ³⁵Cl₂ ¹²C¹H₂, ³⁵Cl³⁷Cl¹²C¹H₂, and ³⁷Cl₂ ¹²CH₂1H₂ made it possible to estimate the fractionation of chlorine isotopes during the bacterial metabolism of DCM. The kinetic chlorine isotope effects for ³⁵Cl³⁷Cl¹²C¹H₂ (m/z 86) and ³⁷Cl₂ ¹²C¹H₂ (m/z 88) relative to ³⁵Cl₂ ¹²C¹H₂ (m/z 84) were characterized by _86/84 = 1.006 ± 0.002 and _88/84 = 1.023 ± 0.003, respectively. The inference is made that the growth of M. dichloromethanicum on DCM is accompanied by the mass-independent fractionation of the DCM isotopomers.     

Detection of two allotype-(Ig-1)-linked minor histocompatibility loci by the use ofH-2-restricted cytotoxic lymphocytes in congenic mice

Cytotoxic lymphocytes (CTL) were generated betweenIg-1-congenic strains BALB/c(H-2^d,Ig-1^a) andC.B-17(H-2^d,Ig-1^b) by cross-immunization in both directions and rechallenge in vitro. The effector cell populations specifically lysed target cells sharing both theH-2 haplotype and theIg-1 allele of the sensitizing strain. B- and T-cell blasts were equally good targets, suggesting thatH-2-restricted cytotoxic lymphocytes are not directed against serologically defined conventional allotypic determinants, but probably against minor histocompatibility antigens controlled by genes linked to theIg-1 complex. Competition experiments using cold target cells from a series ofIg-1^b-congenic strains of the BALB/c background (BAB-14, C.B-17, C.B-26) revealed two not yet described minor histocompatibility loci linked to theIg-1 complex: We could demonstrate that BALB/c anti-C.B-17 effector cells recognize at least two distinct antigenic determinants on C.B-17 target cells, but only one on target cells from BAB-14, which carries a recombinantIg-1 complex. From these results we conclude that one of the minor histocompatibility antigens, designated as H(C_H), is encoded by a gene linked to the heavy-chain constant-region (C_H) genes, whereas the second minor histocompatibility antigen, designated as H(V_H), is coded for by a gene linked to the heavy-chain variable-region (V_H) genes. These two new genetic markers may be useful for further analysis of the mouseIg-1 complex because the analysis of the H(C_H) and H(V_H) genes may facilitate the search for recombinants in that chromosomal region.     

Oxygen-dependent desulphation of monomethyl sulphate by Agrobacterium sp. M3C

Agrobacterium sp. M3C, previously isolated from canal-water for its ability to grow on monomethyl sulphate, degraded this ester with stoichiometric liberation of inorganic sulphate. In contrast with the biodegradation of monomethyl sulphate in Hyphomicrobium sp., and of other longer-chain alkyl sulphates in Pseudomonas spp., the pathway in Agrobacterium appeared not to involve a sulphatase enzyme capable of catalysing ester-bond hydrolysis. No such sulphatase was detectable under a range of conditions of bacterial culture, or using various methods for preparing cell-extracts, or different assay conditions. There was no incorporation of ¹⁸O-label from H₂ ¹⁸O into the liberated inorganic sulphate. No methanol was detectable during biodegradation, and the organism was incapable of growth on methanol, and did not produce methanol dehydrogenase activity when grown on monomethyl sulphate. Tracer studies using mono[¹⁴C]-methyl sulphate indicated that formate serine and glycine were produced during the biodegradation. The presence of these amino acids, together with high activity of hydroxypyruvate reductase, indicated the operation of the serine pathway common in methylotrophs. Use of an oxygen electrode in conjunction with monomethyl[³⁵S]sulphate showed that release of ³⁵SO₄ ^2- was dependent on availability of O₂, and that there was equimolar stoichiometry among monomethyl sulphate degraded, O₂ consumed and ³⁵SO₄ ^2- released. A proposed pathway for the degradation involved an initial mono-oxygenation to methanediol monosulphate with subsequent elimination of SO₄ ^2- and concomitant formation of formaldehyde. The pathway was compared with degradation mechanisms for other C₁ compounds and for other sulphate esters.     

Utilization of Methane and Carbon Dioxide by Symbiotrophic Bacteria in Gills of Mytilidae (Bathymodiolus) from the Rainbow and Logachev Hydrothermal Fields on the Mid-Atlantic Ridge

Bivalve mollusks Bathymodiolus asoricus and Bathymodiolus puteoserpentis collected from the Rainbow and Logachev hydrothermal fields during dives of the Mir 1 and Mir 2 deep-sea manned submersibles were studied. Rates of methane oxidation and carbon dioxide assimilation in mussel gill tissue were determined by radiolabel analysis. During oxidation of ¹⁴CH₄, radiocarbon was detected in significant quantities not only in carbon dioxide but also in dissolved organic matter, most notably ¹⁴C-formate and ¹⁴C-acetate, occurring in a 2 : 1 ratio. Activities of hexulose-phosphate synthase, phosphoribulokinase, and ribulose 1,5-bisphosphate carboxylase were shown in the soluble fraction of gill tissue cells. At the same time, no activity of hydroxypyruvate reductase—the key enzyme of the serine pathway of C₁-assimilation—was detected. The results of PCR amplification using genetic probes for membrane-bound methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF) attest to the presence of the genes of these enzymes in the total DNA extracted from gill samples. However, no appropriate PCR responses were obtained with the mmoX primer system, which is a marker for soluble methane monooxygenase. All samples studied showed amplification with primers for the genera Methylobacter and Methylosphaera. At the same time, no genes specific to the genera Methylomonas, Methylococcus, Methylomicrobium, or MethylosinusandMethylocystis were detected. Electron microscopic examinations revealed the presence of two groups of endosymbiotic bacteria in the mussel gill tissue. The first group was represented by large cells possessing a complex system of cytoplasmic membranes, typical of methanotrophs of morphotype I. The other type of endosymbionts, having much smaller cells and lacking intracellular membrane structures, is likely to be constituted by sulfur bacteria.     

Isolation and Structure of Maingayic Acid,a Piscicidal Constituent of Callicarpa maingayi

A piscicidal constituent (1), C₂₀H₂₈O₃, (chloroform), which was named maingayic acid, was isolated from the leaf of Callicarpa maingayi. On the basis of the chemical spectral studies, the pK_MCS evaluation and the octant rule on the ORD curves, we have and deduced that maingayic acid is a furanoid diterpene acid possessing a rearranged labdane skeleton shown as 1’a.