A novel epitope of the LFA-1 antigen which can distinguish killer effector and suppressor cells in human CD8 cells

The CD4 subset of cells displays helper/inducer activity and recognizes class II antigens of the major histocompatibility complex (MHC), while the CD8 subset recognizes class I MHC antigens and exhibits cytotoxic or suppressor function. Considerable functional as well as corresponding phenotypic heterogeneity exists within the two major T cell subsets. Although the CD8+ population contains pre-cytotoxic, cytotoxic, pre-suppressor and suppressor effector T cells, these distinctions still rest largely on the use of functional assays. Attempts have been made to define the CD8+ precursor of the killer cell with new monoclonal antibodies. But more precise phenotypic distinctions between the functional subpopulations within CD8+ cells will be needed. We have now developed a monoclonal antibody, anti-S6F1 which can distinguish killer effector and suppressor effector cells in CD8 lymphocyte populations. The cell-surface structure defined by this antibody comprises two glycoproteins with relative molecular mass (Mr) 180K and 95K respectively. Also sequential immunoprecipitation studies and two dimensional gel electrophoresis indicate that anti-S6F1 recognizes a novel epitope on the LFA-1 antigen.     

Correlation between a DNA restriction fragment length polymorphism and C4A6 protein

The fourth component of complement (C4) in man, is coded for by two separate but closely linked loci (C4A and C4B) within the major histocompatibility region (MHC), on the short arm of chromosome 6. Like class I and II loci of this region, the C4 genes are highly polymorphic with more than 30 alleles, including null alleles, assigned to the two loci. This extensive polymorphism, based mainly on electrophoretic mobility, provides a useful marker for studies of disease susceptibility. Several disorders, including systemic lupus erythematosus and type I diabetes, show associations with C4 phenotypes. We have used the technique of Southern with a C4 specific probe to examine the genomic DNA of individuals typed for C4 by protein electrophoresis. We have identified 10.7 and 3.8 kilobase (kb) BglII restriction fragments in each of 9 unrelated individuals with a C4A6 allele, and in none of 22 unrelated individuals in whom this allele was not expressed. This clear correlation of restriction fragment length polymorphism with C4 phenotype provides a precise basis for analysis of C4 polymorphism. It is likely to be of value in clinical investigations of autoimmune disease.     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

A molecular map of the immune response region from the major histocompatibility complex of the mouse

A stretch of 200 kilobases (kb) of DNA from the I region of the mouse major histocompatibility complex has been cloned and characterized. It contains the genes for the biochemically defined class II proteins E alpha, E beta and A beta. DNA blot analyses suggest that the I region may contain only 6-8 class II genes. Correlation of our molecular map with the genetic map of the I region confines two of the five I subregions, I-J and I-B, to less than 3.4 kb of DNA at the 3' end of the E beta gene where a hotspot for recombination has been observed. Indeed, the I-A and I-E subregions may be contiguous. If so, the I-B and I-J subregions are not encoded in the I region between the I-A and I-E subregions.     

Long-range restriction site mapping of mammalian genomic DNA

Molecular analysis of many problems in genetics would be facilitated by the ability to construct restriction site maps of long stretches of genomic DNA and to directly place genes on these maps. Pulsed-field gradient gel electrophoresis allows measurement of the size of DNA fragments up to at least 2,000 kilobase pairs (kb) long and we have used this technique here to map sites for one class of infrequently cutting restriction enzyme over a total of 1,500 kb of mouse genomic DNA. The sites for these enzymes tend to be clustered in the genome. These clusters may correspond to the short stretches of C + G-rich unmethylated DNA often associated with mammalian genes.     

Long-range restriction map around the Duchenne muscular dystrophy gene

Duchenne muscular dystrophy is an X-linked recessive disease affecting about 1 in 4,000 newborn boys. As in many other inherited diseases, the biochemical basis of the condition is unknown, and as yet there is no effective treatment. Translocations, deletions and other mutations leading to the DMD phenotype are distributed over a chromosomal area of large, but unknown size. Using pulsed-field gradient gel electrophoresis, we have now determined restriction maps of a major fraction of this area, covering two regions of three million basepairs in total, and used it to determine the position of several probes linked to DMD. The maps establish physical distances between structural changes associated with the DMD phenotype and provide evidence for a CpG-rich island proximal to the area containing translocations and deletions associated with the DMD phenotype.     

甜菜高分子量核DNA的分离

研究了从甜菜中分离高分子量核DNA的方法．利用离心分离细胞核，经低融点琼脂糖块包埋、蛋白酶K原位裂解，结合低融点琼脂糖脉冲电泳去除淀粉制备高分子量核DNA．结果表明利用幼叶制备高分子量核DNA分子量高，更容易去除其中的淀粉；并且利用该方法得到的高分子量核DNA纯度高，适于部分酶切．     

Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A

The major histocompatibility complex (MHC) on chromosome 6 is associated with susceptibility to more common diseases than any other region of the human genome, including almost all disorders classified as autoimmune. In type 1 diabetes the major genetic susceptibility determinants have been mapped to the MHC class II genes HLA-DQB1 and HLA-DRB1 (refs 1-3), but these genes cannot completely explain the association between type 1 diabetes and the MHC region. Owing to the region's extreme gene density, the multiplicity of disease-associated alleles, strong associations between alleles, limited genotyping capability, and inadequate statistical approaches and sample sizes, which, and how many, loci within the MHC determine susceptibility remains unclear. Here, in several large type 1 diabetes data sets, we analyse a combined total of 1,729 polymorphisms, and apply statistical methods-recursive partitioning and regression-to pinpoint disease susceptibility to the MHC class I genes HLA-B and HLA-A (risk ratios >1.5; P(combined) = 2.01 x 10(-19) and 2.35 x 10(-13), respectively) in addition to the established associations of the MHC class II genes. Other loci with smaller and/or rarer effects might also be involved, but to find these, future searches must take into account both the HLA class II and class I genes and use even larger samples. Taken together with previous studies, we conclude that MHC-class-I-mediated events, principally involving HLA-B*39, contribute to the aetiology of type 1 diabetes.     

Evidence for the T3-associated 90K heterodimer as the T-cell antigen receptor

Several surface molecules appear to be involved in antigen recognition by human T lymphocytes including the monomorphic 20/25K T3 structure present on all mature T lymphocytes and the subset-specific associative recognition elements, T4 and T8 (refs 1-8). More recently, Ti1, a clonally unique antigen recognition structure comprised of a 49,000 molecular weight (49K) alpha-chain and a 43K beta-chain, linked to T3 was identified on a major histocompatibility complex (MHC) class I specific T8+ T-cell clone, CT8III (ref. 9). To determine whether analogous receptor molecules could be found on other T-cell clones of differing specificity, we produced monoclonal antibodies against a clonal structure (Ti2) on an MHC class II specific T4+ lymphocyte, CT4II, derived from the same donor as CT8III. The Ti2 structure on CT4II is shown here to be a disulphide-linked heterodimer like Ti1 on CT8III and is composed of subunits of similar molecular weight. Monoclonal antibodies against Ti2 or Ti1 block antigen specific functions of the respective clone without showing any cross-reactivity. These findings suggest that each T lymphocyte, regardless of subset derivation or specificity, uses an analogous Ti heterodimer for antigen specific function. The latter is linked to T3 and expressed on the cell surface at an identical density (30,000-40,000 sites per cell).     

Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain

Gelsolin is representative of a class of actin-modulating proteins found in lower eukaryotes to mammals, which sever actin filaments. Gelsolin found in the cytoplasm of cells is functionally similar to a mammalian plasma protein of similar size, originally called ADF or brevin. Human plasma and rabbit macrophage gelsolins differ by the presence of a 25-amino-acid residue extension on plasma gelsolin which appears to account for the difference in relative molecular mass (Mr) between the proteins as assessed by SDS-polyacrylamide gel electrophoresis (PAGE), 93,000 (93K) and 90K, respectively. Here we report the isolation of full-length human plasma gelsolin complementary DNA clones from a HepG2 library. The inferred amino-acid sequence reveals the presence of a signal peptide, a long tandem repeat that matches the actin-binding domains of gelsolin, a tetrapeptide present in actin and extended regions of identical sequence with rabbit macrophage gelsolin. Southern blot analysis indicates that a single gene in the haploid genome encodes both protein forms.     

Subunit of the '20S' proteasome (multicatalytic proteinase) encoded by the major histocompatibility complex

Cytotoxic T lymphocytes recognize fragments (peptides) of protein antigens presented by major histocompatibility complex (MHC) class I molecules. In general, the peptides are derived from cytosolic proteins and are then transported to the endoplasmic reticulum where they assemble with the MHC class I heavy chains and beta 2-microglobulin to form stable and functional class I molecules. The proteases involved in the generation of these peptides are unknown. One candidate is the proteasome, a nonlysosomal proteinase complex abundantly present in the cytosol. Proteasomes have several proteolytically active sites and are complexes of high relative molecular mass (Mr about 600K), consisting of about 20-30 subunits with Mrs between 15 and 30K. Here we show that at least one of these subunits is encoded by the mouse MHC in the region between the K locus and the MHC class II region, and inducible by interferon-gamma. This raises the intriguing possibility that the MHC encodes not only the MHC class I molecules themselves but also proteases involved in the formation of MHC-binding peptides.     

Fission yeast p107wee1 mitotic inhibitor is a tyrosine/serine kinase.

The fission yeast wee1+ gene product is a dose-dependent, negative regulator of entry into mitosis. wee1+ encodes a protein of relative molecular mass 107,000 (Mr 107K), the C-terminal third of which has strong similarities with the serine/threonine protein kinase family. Here we report that p107wee1 immune complexes phosphorylate p107wee1 equally on serine and tyrosine residues, and also phosphorylate an exogenous substrate, angiotensin II, on tyrosine. Both kinase activities are attributable to p107wee1 because they are also observed when wee1+ is expressed in heterologous systems; both are abolished by a point mutation in the ATP-binding domain, and both behave like an asymmetric monomer of Mr114K on gel filtration and density-gradient centrifugation. Thus the wee1+ gene product is representative of a novel class of protein kinase that phosphorylates both serine and tyrosine residues.     

Cloning and expression in Escherichia coli of the gene for human tumour necrosis factor

Tumour necrosis factor (TNF) was found originally in mouse serum after intravenous injection of bacterial endotoxin into mice primed with viable Mycobacterium bovis, strain Bacillus Calmette-Guerin (BCG). TNF-containing serum from mice is cytotoxic or cytostatic to a number of mouse and human transformed cell lines, but less or not toxic to normal cells in vitro. It causes necrosis of transplantable tumours in mice. TNF also occurs in serum of rat, rabbit and guinea pig. Rabbit TNF has been purified recently to give a single band on SDS-polyacrylamide gel electrophoresis (PAGE). The purified TNF had a relative molecular mass (Mr) 40,000 +/- 5,000 measured by gel filtration, and 17,000 by SDS-PAGE. Its isoelectric point is 5.0 +/- 0.3. The necrotic activity in vivo and the cytotoxicity in vitro are produced by the same substance. The gene encoding TNF has been identified in a human genomic DNA library using as a probe a cloned cDNA encoding a portion of rabbit TNF. The regions of this gene encoding an amino-acid sequence corresponding to mature TNF have been expressed in Escherichia coli and the product of this expression isolated in pure form and shown to produce necrosis of murine tumours in vivo.     

从动物血液中提取基因组DNA方法的比较研究

从血液中提取基因组DNA的传统方法是先得到淋巴细胞,再用蛋白酶K、SDS消化,然后用酚、氯仿抽提,乙醇沉淀．CTAB法提取基因组DNA多应用于植物和微生物,从全血中提取基因组DNA报道甚少．采用改良的CTAB法从全血中提取基因组DNA,并以传统方法和TAKARA Blood Genome DNA Extraction Kit作对照．结果表明：CTAB法可以得到大量的、较完整的基因组DNA,并且纯度达到1．8～2．0,可以满足PCR扩增和限制酶切等实验的要求．     

Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites

Recently, a class of protein antigens of high relative molecular mass (Mt) which can induce protective immunity against blood-stage malaria has been identified. In Plasmodium falciparum the protein has a Mr of approximately 195,000 (P195). It is the precursor of three proteins of Mr 83,000 (83K), 42K and 19K which are the major surface antigens of merozoites; thus it may also be useful for immunization against P. falciparum. Three studies describing the isolation of single short complementary DNA clones for part of the P195 gene sequence have been reported. Here we describe the complete structure of the P195 gene determined from further DNA clones, its organization within genomic DNA and the location of the specific processing fragments within the primary amino-acid sequence.     

水解酸化-好氧处理含油污水中微生物群落变化研究

为揭示水解酸化-好氧处理系统处理油田采出水过程中生物群落的变化规律,考察对不同水力停留时间(tHAT)下好氧池的生物相,提取水解酸化池和好氧池中菌体的脱氧核糖核酸(DNA),以细菌通用引物对DNA V3高变异区聚合酶链式反应(PCR)扩增后进行变性梯废凝胶电泳(DGGE)分析.结果表明:当水解酸化池和好氧池的水力停留时间分别大于12和16 h时,镜检有高级原生动物,出水CODCr去除率大于58.73%,含油去除率大于98.61%;水解酸化-好氧处理工艺在不同tHRT条件下运行后,在形成的细菌群落中,既有相同的优势群落,也有各自特有的优势群落;随着tHRT的延长,水解酸化池中优势群落的种类和数量增加,当tHRT＞12 h时,优势群落变化较小,形成稳定的细菌群落,出水水质趋于稳定;随着tHRT的延长,好氧处理池中优势群落的种类和数量减少,当tHRT＞16 h时,优势群落变化较小,形成稳定的生态系统,出水水质趋于最佳.     

太白红杉总DNA的快速提取方法

对太白红杉总DNA提取方法进行了探讨，并对其不同部位和器官、不同处理方式材料进行了提取实验研究．通过琼脂糖凝胶电泳、限制性内切酶处理等方法对所提取的DNA样品进行检测．结果表明，改良后的CTAB法，对于太白红杉总DNA的提取是一种良好的简单易行的方法，无论是烘干后的材料还是新鲜材料提取的总DNA中基本没有蛋白质及酚类污染．该方法可在短时间内获得大量的DNA样品，并可根据需要对样品进行适当操作．     

不同方法提取水溞基因组DNA的比较

为获取抽提水溞基因组DNA的最适方法,本研究采用酚-氯仿法,CTAB法和试剂盒法3种方法提取水溞基因组DNA,并用琼脂糖凝胶电泳检测DNA提取的效果.结果表明3种DNA提取方法均获得较好的DNA,其中试剂盒方法提取的DNA最为理想,完全可满足后续分子生物学实验.     

Novel submicroscopic extrachromosomal elements containing amplified genes in human cells

In previous work, several methotrexate (MTX)-resistant variants were isolated frm the human cell line HeLa BU25, which exhibited a high degree of dihydrofolate (DHFR) gene amplification (estimated to be 250- to 300-fold). These variants did not contain any chromosome with a homogeneously staining region (HSR) and exhibited only a small average number of minute chromosomes per cell: these two types of karyotypic abnormalities generally accompany selective gene amplification. We now report that structures containing amplified DHFR genes in one of these variants (HeLa BU25-10B3) can be isolated by pulsed-field gradient or field-inversion gel electrophoresis as homogeneous DNA molecules of approximately 650 kilobases (kb). Electron microscopy of metaphase spreads from these cells reveals chromatin fibres with a similar DNA content, which are probably related to the above elements. These represent a novel type of extrachromosomal structures in mammalian cells.     

Characterization of an expressed CD3-associated Ti gamma-chain reveals C gamma domain polymorphism

The majority of human T cells express an antigen receptor consisting of a disulphide-linked heterodimer (Ti) of relative molecular mass 80,000-90,000 (Mr 80-90K) which is noncovalently associated with a set of at least three proteins of Mr 20-28K termed CD3 (Leu4, T3). Whereas both chains of Ti, an acidic alpha-chain of Mr 48-54K and a more basic beta-chain of Mr 40-44K, contain variable and constant region domains, the component peptides of CD3 are invariant. Several laboratories have more recently reported the expression of CD3 in association with a novel protein. On the surface of long-term T-cell lines and one thymocyte clone this novel structure consists of a 40K protein noncovalently linked to a 55 or 62K protein identified as the protein product of the Ti gamma-chain gene, a T-cell specific gene which like the Ti alpha- and Ti beta-chain genes undergoes rearrangement of variable (V) and joining (J) region gene segments. On the human T-cell leukaemic line PEER we have detected only a single 55K glycoprotein associated with CD3. We here demonstrate that an anti-Ti gamma-peptide antiserum reacts with the 55K CD3-associated protein on PEER. Most previously described human Ti gamma-chain complementary DNA clones encode the products of non-functional rearrangements. One of the Ti gamma cDNAs isolated from PEER, however, represents a functional rearrangement reported for the first time in a cell which expresses a Ti gamma-chain protein product on the cell surface. Interestingly, a 48-base-pair (bp) sequence in the constant (C) region domain of this functional Ti gamma-chain cDNA is triplicated in PEER and duplicated in other cDNAs isolated from PEER and other cell lines.