非放射性反相杂交法在HLA－DQA1位点基因分型中的应用

将遴选的经适当接尾的１２个ＨＬＡ－ＤＱＡ１序列特异性寡核苷酸固定在一张滤膜上,用生物素标记的ＤＱＡ１特异性扩增产物与滤膜上的序列特异性寡核苷酸在四甲基氯化铵杂交体系中杂交,然后经洗膜封膜,杂交信号用非放射性的碱性磷酸酶显色法检测,根据杂交斑点的显示结果分析标本的基因型。采用这种方法初步确定了ＨＬＡ－ＤＱＡ１位点８种单倍型等位基因：ＤＱＡ１０１０１、０１０２、０１０３、０２０１、０３０１１、０４０１、０５０１和０６０１．非放射性反相杂交法可对各种来源的杂合性标本进行ＨＬＡ－Ⅱ类基因快速分型,并适合在临床器官移植的组织分型配型、疾病易感性研究和法医鉴定等领城中应用。     

Oligozymes

The simple use of nonisotopic hybridization probes to detect complementary sequences provides valuable information in a large number of research and commercial applications. In hybridization assays, the four ‘S’s (speed, simplicity, sensitivity, and specificity) are important criteria for determining the choice of probe and label. The direct chemical combination of synthetic oligonucleotide probes and enzyme labels offer advantages unmatched by other approaches, with the oligonucleotide providing rapid hybridization and high specificity, and the direct enzyme label providing simple and sensitive detection. Such oligonucleotide-enzyme conjugates (“oligozymes”) can be used in a variety of hybridization and detection formats, including dot blots, Southern/northern blots,in situ, and solution hybridization/capture schemes. The practical synthesis and use of such oligozymes are summarized.     

The Chromosomal Organization of the Human Endogenous Retrovirus-like Sequence HERV-H: Clustering of the HERV-H Sequences in a 300-kb Region Close to the GRPR Locus on the X Chromosome

Within the haploid genome there are approximately 1,000 copiesof the human endogenous retroviruslike sequence, HERV-H. Althoughthese sequences are scattered throughout the entire genome,in situ hybridization experiments revealed that there are discreteclusters positioned on chromosomes 1p and 7q. In this study,we have located three HERV-H sequences which were unexpectedlyclustered within a 300-kilobase region close to the GRPR locuson the X chromosome. In previous studies, no clusteringof thissequence has been reported at this locus. Our finding demonstratesthat, like other repetitive sequences, clustering of HERV-Hoccurs in the human genome, although these sequences may notalways be detected by in situ hybridization methods.     

Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

Isolation,characterization and application of a species-specific repeated sequence from Haynaldia villosa

A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations.     

Identification of New Oligodendrocyte- and Myelin-Specific Genes by a Differential Screening Approach

Abstract: We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte-free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide-synthesizing enzyme UDP-galactose:ceramide galactosyl-transferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T-lymphocyte-specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl-CoA-desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long-chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the β-tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.     

A second L-type isozyme of potato glucan phosphorylase: cloning,antisense inhibition and expression analysis

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81–84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1–81) and the insertion sequence (amino acids 463–570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively.Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.