A conserved sequence in the immunoglobulin J kappa-C kappa intron: possible enhancer element

Several functionally important genetic elements (such as the TATA box, mRNA splice sequences, poly(A) addition signal) were first detected as short segments of unexplained sequence homology within non-coding regions of different genes. A short region of unknown sequence in the intron between the human J kappa and C kappa immunoglobulin coding regions was found to be sufficiently homologous to the corresponding segment of the mouse gene to form stable heteroduplexes. Although no specific function has yet been definitely ascribed to this region (which we call the kappa intron conserved region, or KICR), some functional significance has been inferred from the findings that (1) activation of B lymphocytes induces a DNase hypersensitivity site in this region and (2) deletions including this region reduce expression of kappa genes introduced into lymphoid cells. To delineate the KICR more precisely and to test the generality of the sequence conservation in a third species, we have sequenced this region of the human and mouse genes and have examined the corresponding region of a recently cloned rabbit kappa gene. We find a segment of about 130 base pairs (bp) that shows striking conservation in all three species, demonstrating homology significantly higher than within the C kappa coding region itself. Two short sequences from the J kappa-C kappa intron that were noted by other investigators to be homologous to proposed 'enhancer' sequences both lie within the conserved region.     

马铃薯卷叶病毒基因组保守序列片段的RT-PCR扩增

从感染马铃薯卷叶病毒的植株中提取马铃薯卷叶病毒总RNA,针对马铃薯卷叶病毒基因组,其中的2个保守序列分别设计并合成了1对寡聚核苷酸引物,用反转录-聚合酶链式反应方法从提取的病毒RNA材料中扩增出符合设计大小的240bp、400bp的特异性产物,对照的健康植株中未扩增出相应产物。     

Somatic hypermutation of an immunoglobulin transgene in kappa transgenic mice

Initial studies of somatically acquired mutations in immunoglobulin V regions from hybridomas and myelomas that are not derived from joining aberrations, suggested a controlled and specific hypermutation process, because spontaneous mutation rates observed for other genes are extremely low. Some evidence for the idea that mutations are introduced during V-gene rearrangement came from the clustering of mutations at the joining sites, from the absence of mutations in unrearranged V genes and from the low level of mutations in only partially (D-J) rearranged nonproductive heavy-chain alleles. Another model in which mutations accumulate with each cell division, rather than being introduced all at once, was supported by the finding that immunoglobulin genes of hybridomas derived from a single mouse frequently had several mutations in common, and so might be derived from the same precursor cell whose daughters then accumulated additional mutations. But the common mutations in some cases could be due to as yet unidentified related germline genes, or could represent the effect of antigen selection for certain amino acids. To try to detect hypermutation in the absence of V-gene rearrangement, we isolated B lymphocytes with endogenous heavy-chain gene mutations from transgenic mice carrying pre-rearranged kappa-transgenes. We found that these kappa-transgenes were also somatically mutated. This and other observations indicated that: ongoing rearrangement is not required for mutation; there are signals for hypermutation in the transgenes; the mutations are found only in the variable region, so the constant region may not be a target; different transgene insertion sites are compatible with hypermutations and more than one transgene is expressed in the same cell.     

A protein binding to the J kappa recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif

Site-specific recombination requires conserved DNA sequences specific to each system, and system-specific proteins that recognize specific DNA sequences. The site-specific recombinases seem to fall into at least two families, based on their protein structure and chemistry of strand breakage. One of these is the resolvase-invertase family, members of which seem to form a serine-phosphate linkage with DNA. Members of the other family, called the integrase family, contain a conserved tyrosine residue that forms a covalent linkage with the 3'-phosphate of DNA at the site of recombination. Structural comparison of integrases shows that these proteins share a highly conserved 40-residue motif. V-(D)-J recombination of the immunoglobulin gene requires conserved recombination signal sequences (RS) of a heptamer CACTGTG and a T-rich nonamer GGTTTTTGT, which are separated by a spacer sequence of either 12 or 23 bases We have recently purified, almost to homogeneity, a protein that specifically binds to the immunoglobulin J kappa RS containing the 23-base-pair spacer sequence. By synthesizing probes on the basis of partial amino-acid sequences of the purified protein, we have now isolated and characterized the complementary DNA of this protein. The amino-acid sequence deduced from the cDNA sequence reveals that the J kappa RS-binding protein has a sequence similar to the 40-residue motif of integrases of phages, bacteria and yeast, indicating that this protein could be involved in V-(D)-J recombination as a recombinase.     

Complete nucleotide sequence of an immunoglobulin VH gene homologue from Caiman, a phylogenetically ancient reptile

Immunoglobulin variable (V) gene regions typify extensive multigenic families in terms of overall size, chromosomal arrangement and presence of large numbers of apparent pseudogenes. A unique mechanism of somatic reorganization involving recombination of VH, D and JH or VL and JL segments accompanies the differentiation of lymphoid cells and together with somatic mutation and other types of recombination accounts for V-region diversity. Although these processes have been well characterized in higher mammals, little is known concerning their origin and diversification during phylogenetic time. Previously, we described the blot-hybridization characteristics of murine VHIII probes with restriction enzyme-digested genomic DNA isolated from several phylogenetically critical species, including Caiman crocodylus, a modern representative of an ancient reptilian subclass. Here we have used a murine probe, S107V, to select homologous clones from a library of Caiman genomic DNA constructed in a lambda bacteriophage. The complete nucleotide sequence of a Caiman gene homologous to the murine VH gene and its adjacent 5' and 3' region is described. Comparison of the sequence with mammalian prototypes shows evidence of considerable organizational and structural homology extending outside the presumed VH-coding region and including elements believed to be involved in somatic recombination. Inferences about the evolution of this multigenic family can now be extended to the level of phylogenetic class.     

蛋白酶结构基因及其克隆

为探明重要毒力因子玫烟色棒束孢几丁质酶基因Ifupr1的表达调控机理,以Ifupr1基因组全长核苷酸序列为基础,采用基因组步移方法,获得Ifupr1的5′-上游区序列(总长为2402bp)。分析其结构基因中无内含子,该序列含有启动子的核心结构序列CAAT框、特异的反应元件和转录因子结合位点。CAAT框是调控转录速率的元件,而其他一些转录因子结合位点则可能具有特异性调节基因转录的功能。     

Identification and comparison of two sequence elements that confer cell-type specific transcription in yeast

The MAT alpha 2 protein of budding yeast represses a set of genes; if the MATa1 protein is also present, a further set of genes is repressed. DNA sequence comparisons reveal a 20-base pair 'operator' sequence that is present in genes repressed by a1/alpha 2. A related, but distinct, sequence is found in genes repressed by alpha 2 alone.     

牦牛催乳素受体基因部分片段的克隆与序列分析

扩增出牦牛催乳素受体（尸碰尺）基因部分保守序列,为研究牦牛乳腺组织中朋三尺基因表达水平奠定基础．提取牦牛乳腺组织总RNA,根据NCB1的奶牛Jp月￡只基因cDNA序列的保守区设计特异性引物,采用RT-PCR技术扩增牦牛尸胜尺基N,结果获得577bp的片段,将该片段连接于pMD18-Simple质粒中,转化大肠杆菌,菌液PCR鉴定阳性克隆子,测序并分析氨基酸序列．分析序列与氨基酸与其他物种同源性,结果表明该序列与水牛、奶牛、绵羊、人、小鼠的尸RLR基因mRNA的对应序列的同源性分别为97．40％、99．13％、93．41％、71．07％、60．20％,编码的氨基酸同源性分别为97．40％、100％、95．83％、82-38％、72-22％．     

Variant (6 ; 15) translocation in a murine plasmacytoma occurs near an immunoglobulin kappa gene but far from the myc oncogene

Chromosome translocations in B-lymphoid tumours are providing intriguing insights and puzzles regarding the role of immunoglobulin genes in the activation of the myc oncogene (reviewed in refs 1, 2). The 15 ; 12 translocations found in most murine plasmacytomas and the analogous 8 ; 14 translocation in human Burkitt's lymphomas involve scissions of murine chromosome 15 (human chromosome 8) near the 5' end of the c-myc gene and subsequent fusion near an immunoglobulin heavy-chain gene. The less well characterized 'variant' translocations found in about 15% of such tumours also involve the myc-bearing chromosome band, but exchange occurs with a chromosome bearing an immunoglobulin light-chain locus--in mice, the kappa-chain locus bearing chromosome 6 (refs 3-5) and, in man, chromosome 2 (or 22), at the same band at which the kappa (or lambda) locus lies (reviewed in ref. 1). The Burkitt variant translocations involve scissions 3' of c-myc; one 8 ; 22 translocation placed the C lambda locus just 3' of c-myc, but usually the chromosome 8 breakpoint is a greater, but unknown, distance away from c-myc, more than 20 kilobases (kb) in one 8 ; 2 translocation involving the C kappa gene. Little is known about the murine 6 ; 15 translocations, although a C kappa gene cloned from one plasmacytoma (PC7183) is linked, via chromosome 12 sequences, to an unidentified region of chromosome 15 (ref. 11). We describe here the chromosome fusion region from plasmacytoma ABPC4, which displays the typical reciprocal 6;15 translocations. We find that the chromosome 6 breakpoint is near C kappa but, unlike those in the heavy-chain locus, not at a position where immunoglobulin genes normally recombine. Moreover, the chromosome 15 sequences involved in the ABPC4 translocation are not derived from the vicinity of c-myc.     

Tetramerization of an RNA oligonucleotide containing a GGGG sequence.

Poly rG can form four-stranded helices. The Hoogsteen-paired quartets of G residues on which such structures depend are so stable that they will form in 5'-GMP solutions, provided that Na+ or K+ are present (see for example, refs 2-4). Telomeric DNA sequences, which are G-rich, adopt four-stranded antiparallel G-quartet conformations in vitro, and parallel tetramerization of G-rich sequences may be involved in meiosis. Here we show that RNAs containing short runs of Gs can also tetramerize. A 19-base oligonucleotide derived from the 5S RNA of Escherichia coli (strand III), 5'GCCGAUGGUAGUGUGGGGU3', forms a K(+)-stabilized tetrameric aggregate that depends on the G residues at its 3' end. This complex is so stable that it would be surprising if similar structures do not occur in nature.     

Construction of an improved baculovirus insecticide containing an insect-specific toxin gene

Baculoviruses provide alternatives to chemicals for controlling insect pests and can be applied by spraying. Baculoviruses have a limited host range, but work relatively slowly. They are dissolved in the midgut of insect larvae to release infectious virions which enter gut epithelial cells and begin to replicate. Replication in other organs causes extensive tissue damage and eventually death. This process can take 4-5 days, but in the field may last for more than a week, allowing the larvae to feed for longer and thereby damaging the host plant. Baculovirus expression vectors expressing foreign genes, such as those for insect-specific toxins, hormones or enzymes, might alleviate this problem. We have now constructed a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus containing an insect-specific neurotoxin from the venom of the North African (Algerian) scorpion, Androctonus australis Hector. The neurotoxin acts by causing specific modifications to the Na+ conductance of neurons, producing a presynaptic excitatory effect leading to paralysis and death; it has no effect in mice. Expression of the neurotoxin by the virus causes a reduction in the time required to kill the host insect.     

Inhibition of the Prausnitz-Küstner reaction by an immunoglobulin epsilon-chain fragment synthesized in E. coli

The Prausnitz-Küstner (P-K) reaction is a sensitive test for the presence and activity in the skin of immunoglobulin E, an important class of immunoglobulin mediating allergic reactions. A fragment of the human myeloma ND epsilon-chain gene, encoding the second, third and fourth domains of the IgE constant region (C epsilon 2-4) was assessed here for its ability to inhibit the P-K reaction in vivo. Injection of the fragment in skin sites of healthy human adults prevented subsequent sensitization with serum containing IgE antibody to ragweed antigen. Inhibition of the P-K reaction required a 200-fold molar excess of the C epsilon fragment over the IgE present in the sensitizing serum. The efficacy of the C epsilon fragment in inhibiting the P-K reaction compared favourably with that of natural myeloma IgE (PS) in terms of both blocking concentrations and duration of the blocking effect. The inhibition of the P-K reaction by C epsilon 2-4 fragments was specific and probably caused by the saturation of IgE receptors on mast cells by the recombinant gene product.