Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification

We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.     

Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1

Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-K) shows that exon-intron boundaries are well conserved between these two species.Accession number of the sequence: X75598     

富亮氨酸重复超家族新成员LRRC4的克隆与在脑瘤中的表达分析

在染色体7q31-32多种肿瘤杂合性丢失（loss of heterozygosity,LOH）高频区,采用表达序列标签（expressed sequence tag,EST）介导的定位候选克隆策略获得了一个定位于人染色体7q31-32的新基因（GenBank 登录号: AF196976）.该基因编码653个氨基酸,蛋白质理论pI/m:6.58/72.7 ku.它包含七个典型的LRR、一个IgC2样结构域.此外,它还包含一个N端信号肽、一个C端跨膜区.其结构特征表明它是富亮氨酸重复（leucine-rich repeat,LRR）超家族的新成员.经过人类基因组命名委员会的同意,将该基因命名为LRRC4.此外,通过序列相似性匹配还获得了定位于小鼠6号染色体的LRRC4的同源基因（GenBank 登录号: AF290542）.RNA印迹和RT-PCR检测发现LRRC4在正常人脑组织相对特异表达,而在多种原发性脑瘤表达明显下调或缺失.综合考虑LRRC4基因的序列特征及表达谱,提示LRRC4基因可能在神经系统中发挥重要作用.     

A sequence with homology to human HPFH-linked enhancer elements and to a family of G-protein linked membrane receptor genes is located downstream of the chicken beta-globin locus.

We report 5805bp of novel sequence (GenBank/EMBL Accession No. AJ012570) from a region starting approx. 11.5kb downstream of the chicken beta-globin locus (map position approx. +30.8 to +36.6kb), which contains a 945bp open reading frame (map position approx. +33 to +33.9kb). This is predicted to encode a 315-residue protein containing seven hydrophobic helical regions and a 17 amino acid motif characteristic of the R7G family of G-protein coupled membrane-bound receptors. The open reading frame and some surrounding sequence also have significant homology with the breakpoint enhancer elements, which also contain open reading frames, implicated in the HPFH-1/2 and HPFH-6 deletional forms of the human syndrome, hereditary persistence of foetal haemoglobin (HPFH). The existence of similar sequences at similar distances downstream of the beta-globin genes in chickens and HPFH patients is intriguing.     

Producing single-stranded DNA probes with the Taq DNA polymerase: a high yield protocol

We report an efficient procedure to synthesize either single- or double-stranded probes labeled with biotin-11-dUTP, biotin-21-dUTP or digoxigenin-11-dUTP. To produce the single-stranded probe, only a single primer is utilized in a Taq polymerase amplification of 55 cycles. A cytomegalovirus probe is presented. This procedure allows easy production of nonradioactively labeled pure single-stranded probes of any desired length and specificity.     

定位于染色体9p21-22的一个新基因UBAP1的克隆测序及在鼻咽癌中的表达分析

 在染色体 9p2 1 2 2鼻咽癌杂合性丢失 (lossofheterozygosity,LOH)高频区 ,应用EST介导的定位 侯选克隆策略 ,用RT PCR及Northern杂交检测了 2 2个表达序列标记 (expressedsequencetag ,EST)在鼻咽癌细胞株HNE1和原代培养的正常鼻咽上皮细胞中的表达差异 ,并对其中一个在鼻咽癌细胞株HNE1中表达下调的EST检测了在鼻咽癌活检组织中的表达 .用生物信息学方法获得其全长cDNA序列 ,GenBank登录号AF2 2 2 0 4 3.该基因cDNA全长 2 70 1bp ,其开放阅读框 (openreadingframe ,ORF)编码一个含 50 2个氨基酸、分子量为 55kD的碱性蛋白质 ,在蛋白羧基端含有 2个连续的重要UBA功能域 (ubiquitinassociateddomain) ,属于遍在蛋白相关蛋白家族的一个新成员 ,经国际人类基因命名委员会同意 ,将其命名为UBAP1 (ubiquitinassociatedprotein 1 ) .Northern表达分析显示UBAP1在所检测的人组织中广泛表达 ,但在人的心脏、骨骼肌及肝脏中的表达较强 .UBAP1基因在63 2 % ( 1 2 1 9)的鼻咽癌活检组织中表达下调 .UBAP1基因作为一个遍在蛋白相关蛋白家族的新成员 ,结合其在 9p的重要定位信息 ,有必要进一步研究其表达下调参与鼻咽癌发生发展的可能机制 .     

Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1

The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S. cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.     

中外两品种鸡胸肌组织差异表达基因的研究

运用mRNA差异显示技术对北京油鸡和AA肉鸡胸肌组织基因的差异表达进行研究, 从分子水平分析导致两品种肌肉组织基因差异表达的机制。通过反向Northern dot blot技术验证共筛选出差异表达基因7条, 经与GenBank数据库进行相似性比对, S1与HMGN3基因有较高同源性; S3与ChEST294a8有很高同源性, 但功能未知; S4和S5与鸡的磷酸葡萄糖变位酶Ⅰ同源性很高; S6和S2与已有核酸数据库中的基因克隆或EST具有较高同源性, 为已知的EST, 但功能未知; 序列S7未在数据中发现同源序列, 可以确定其在AA肉鸡中特异表达,确定为新发现的EST(GenBank登录号: EU594549)。为进一步研究中外两品种鸡胸肌组织基因差异表达机制奠定了基础。     

A revision of the human XIST gene organization and structural comparison with mouse Xist

The XIST gene plays an essential role in X Chromosome (Chr) inactivation during the early development of female humans. It is believed that the XIST gene, not encoding a protein, functions as an RNA. The XIST cDNA is unusually long, as its full length is reported to be 16.5 kilobase pairs (kb). Here, comparison of sequences from the genomic interval downstream to the 3′ end of the human XIST gene against the human EST database brought to light a number of human EST sequences that are mapped to the region. Furthermore, PCR amplification of human cDNA libraries and RNA fluorescence in situ hybridization (RNA-FISH) demonstrate that the human XIST gene has additional 2.8 kb downstream sequences which have not been documented as a part of the gene. These data show that the full-length XIST cDNA is, in fact, 19.3 kb, not 16.5 kb as previously reported. The newly defined region contains an intron that may be alternatively spliced and seven polyadenylation signal sequences. Sequences in the newly defined region show overall sequence similarity with the 3′ terminal region of mouse Xist, and three subregions exhibit quite high sequence conservation. Interestingly, the new intron spans the first two subregions that are absent in one of the two isoforms of mouse Xist. Taken together, we revise the structure of human XIST cDNA and compare cDNA structures between human and mouse XIST/Xist. Received: 3 August 1999 / Accepted: 15 November 1999     

Cloning of thrombolytic enzyme (lumbrokinase) from earthworm and its expression in the yeast Pichia pastoris

Lumbrokinase (PI239, GenBank Accession No. AF433650) from the earthworm Lumbricus bimastus has been identified. The cDNA of PI239 is composed of 852bp and includes an open reading frame that encodes two parts of the protein: a signal peptide of 44 amino acids and a mature peptide of 239 residues. The cDNA of PI239 exhibits a high degree of sequence identity with other lumbrokinase genes, ranging from 87.6% (F-III-I) to 98.3% (EFE-3). The gene encoding the native form of PI239, with a 5' non-functional end removed, was obtained by PCR amplification and was sub-cloned into pPICZalpha-A, a yeast expression and secretion vector. SDS-PAGE and Western blot analyses showed that rPI239 secreted into the culture medium was specifically recognized by the wild type lumbrokinase polyclonal antibody and was able to dissolve artificial fibrin plates.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

人肾高亲和力钠离子依赖性二羧酸转运蛋白基因的克隆和生物信息学分析

为探讨人高亲和力钠离子依赖性二羧酸转运蛋白 (humanhigh affinitysodium dependentdicar boxylatetransporter,hSDCT2orhNaDC3 )基因在人体内的生理功能及其与疾病的关系 ,借助生物信息学成功地从人肾中克隆了hSDCT2基因 (GenBank接收号 :AY0 72 810 ) .首先将大鼠SDCT2cDNA与人EST数据库进行同源性比较 ,获得具有高度同源性EST片段并用DNAstar软件将它们拼接成EST重叠群 .在重叠群上设计PCR引物从人肾总RNA中用RT PCR扩增出hSDCT2基因并测序 ,然后用软件对其结构特性、组织分布及基因定位进行分析 .序列测定结果显示 ,hSDCT2开放阅读框为180 9bp ,共编码 6 0 2个氨基酸 .蛋白同源性分析表明 ,其氨基酸序列与大鼠及小鼠SDCT2分别有85 %和 87%相同 .二级结构分析显示 ,该蛋白有 12个跨膜螺旋区 .Northern分析显示 ,该基因可在肾、肝、脑、胎盘等多种组织中表达 ,并定位于 2 0号染色体的q12～q13 1     

In Situ Detection of a PCR-Synthesized Human Pancentromeric DNA Hybridization Probe by Color Pigment Immunostaining: Application for Dicentric Assay Automation

We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.