Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes.

The B1 (CD20) molecule is a Mr 33,000 phosphoprotein on the surface of human B lymphocytes that may serve a central role in the humoral immune response by regulating B-cell proliferation and differentiation. In this report, a cDNA clone that encodes the B1 molecule was isolated and the amino acid sequence of B1 was determined. B-cell-specific cDNA clones were selected from a human tonsillar cDNA library by differential hybridization with labeled cDNA derived from either size-fractionated B-cell mRNA or size-fractionated T-cell mRNA. Of the 261 cDNA clones isolated, 3 cross-hybridizing cDNA clones were chosen as potential candidates for encoding B1 based on their selective hybridization to RNA from B1-positive cell lines. The longest clone, pB1-21, contained a 2.8-kilobase insert with an 891-base-pair open reading frame that encodes a protein of 33 kDa. mRNA synthesized from the pB1-21 cDNA clone in vitro was translated into a protein of the same apparent molecular weight as B1. Limited proteinase digestion of the pB1-21 translation product and B1 generated peptides of the same sizes, indicating that the pB1-21 cDNA encodes the B1 molecule. Gel blot analysis indicated that pB1-21 hybridized with two mRNA species of 2.8 and 3.4 kilobases only in B1-positive cell lines. The amino acid sequence deduced from the pB1-21 nucleotide sequence apparently lacks a signal sequence and contains three extensive hydrophobic regions. The deduced B1 amino acid sequence shows no significant homology with other known proteins.     

Cloning of the chicken progesterone receptor.

Monospecific antibodies directed against the chicken progesterone receptor (PR) form B were used to screen a randomly primed phage lambda gt11 cDNA expression library prepared from size-fractionated chicken oviduct mRNA. Two independent immunoreactive clones, lambda cPR1 and lambda cPR2, were isolated. Antibodies selected from anti-PR form B antiserum on matrices of lambda cPR1 and lambda cPR2 fusion proteins detected two proteins on electrophoretic immunoblots of crude and purified PR preparations. These proteins had the same apparent molecular weights as did PR forms A and B crosslinked with the tritiated progestin R 5020. Thus, lambda cPR1 and lambda cPR2 fusion proteins contain epitopes present in both PR forms A and B. A cDNA clone, lambda cPR3, containing the inserts of both lambda cPR1 and lambda cPR2, was isolated from a randomly primed lambda gt10 oviduct cDNA library, indicating that both cDNA inserts were derived from the same oviduct mRNA. Additional evidence that these cDNAs correspond to PR mRNA was provided by sequencing the lambda cPR3 cDNA insert, since it was found to encode the sequence of three tryptic peptides prepared from purified PR form B. A fourth and a fifth cDNA clone, lambda cPR4 and lambda cPR5, were sequentially isolated from the same lambda gt10 cDNA library beginning with a probe derived from the 3' end of the lambda cPR3 insert. Partial DNA sequencing of lambda cPR4 and lambda cPR5 revealed the presence of a sequence coding for a cysteine-rich domain that is strikingly homologous to the amino acid sequences present in the putative DNA-binding domain of the human and chicken estrogen receptors, human glucocorticoid receptor, and v-erbA gene product of the avian erythroblastosis virus.     

Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequences, the carboxyl-terminal peptide and the 3'-untranslated region are highly divergent and specific for this cDNA clone. There appears to be an amino acid deletion in the 3' end of the hamster alpha myosin heavy chain as compared to the rat alpha myosin heavy chain. S1 nuclease mapping experiments have shown that the mRNA represented by this cDNA clone is scarcely expressed in neonatal development, but its expression increases with age and reaches maximal levels in adult life. This cDNA clone provides a useful tool to follow the myosin heavy chain mRNA changes during development and during the genesis of a cardiomyopathy, an autosomal recessive defect carried by the Syrian hamster.     

应用抑制性消减杂交技术克隆和筛选PS1TP5反式激活相关基因

目的构建乙型肝炎病毒前-S1蛋白反式激活基因5(PS1TP5)的反式激活相关基因差异表达的cDNA,克隆PS1TP5蛋白反式激活相关基因。方法以PS1TP5表达质粒pcDNA3.1(-)-myc-his(A)-PS1TP5转染HepG2细胞,以空载体pcDNA3.1(-)-myc-his(A)为对照,制备转染后的细胞裂解液,提取mRNA并合成cDNA,经RsaⅠ酶切后将实验组cDNA分成两组,分别衔接两种不同接头,再与对照组cDNA进行两次消减杂交及两次PCR反应,产物与T/A克隆载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆经PCR鉴定后进行测序及同源性分析。结果成功构建人PS1TP5蛋白反式激活相关基因差异表达的cDNA。扩增后得到70个200～1 000 bp插入片段的克隆,随机挑选其中30个插入片段测序,并通过生物信息学分析获得其全长基因序列,结果共获得24种编码基因,其中1个为未知功能的新基因,电子拼接后命名为HBV PS1TP5TP1(乙型肝炎病毒前-S1蛋白反式激活蛋白5反式激活蛋白1),GenBank注册号DQ487761。结论筛选到的cDNA全长序列,包括一些与细胞...     

Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences.     

Sequence of a second human asialoglycoprotein receptor: conservation of two receptor genes during evolution.

The asialoglycoprotein (ASGP) receptor isolated from human liver and from the human hepatoma cell line HepG2 migrates on NaDodSO4 gel electrophoresis as a single species of 45,000 daltons. Recently, we isolated a cDNA clone encoding this receptor (H1) from a HepG2 lambda gt11 library. From the same library, we have isolated and sequenced a clone encoding a second ASGP receptor, H2, with a protein sequence homology of 58% to H1. There are two subspecies of H2 that differ only by the presence of a five-amino acid insertion in the COOH-terminal extracytoplasmic domain. Comparison with the available sequences of the two rat ASGP receptors R1 and R2 indicates that H1 is more homologous to R1 than to H2, and H2 is more similar to R2 than to H1. Thus, the two receptor genes evolved before the separation of rat and man. As judged by RNA blot hybridization of HepG2 RNA using RNA transcribed in vitro from cDNA clones of the human receptors as standards, H1 and H2 mRNA are present in equimolar amounts, each 0.005-0.01% of the total mRNA. This finding raises the question of whether the three ASGP receptor proteins are functional as heterodimers or whether they might serve different functions in the cell.     

Estrogen regulation of alpha 1(I)-procollagen messenger ribonucleic acid in the rat uterus

A cDNA library, prepared from mRNA isolated from the uteri of 3-day estradiol-stimulated immature rats, was constructed in pBR322. From this library an estrogen-regulated clone, pERU3, was isolated. This clone contained sequences complementary to uterine mRNA that migrated during gel electrophoresis as a double band of about 5.0 and 5.8 kilobases. Little of this mRNA was seen in several other tissues examined. An increase in the amount of this RNA in uterus was seen 2 h after estradiol treatment, with maximum hybridization occurring, in different experiments, between 18 and 36 h, followed by a decline. Hybridization of the cDNA insert of the pERU3 plasmid with known probes indicated that it coded for alpha 1(I)-procollagen. This conclusion was supported by in vitro translation experiments in which the hybrid-selected mRNA complementary to pERU3 DNA was shown to code for a collagenase-sensitive protein with a size corresponding to that of alpha 1(I)-procollagen. This system, therefore, provides an additional tool for the study of the estrogen regulation of gene expression in the uterus.     

Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver.

Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library [Battini, R., Ferrari, S., Kaczmarek, L., Calabretta, B., Chen, S. & Baserga, R. (1987) J. Biol. Chem. 262, 4355-4359], with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH2-end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones.     

Isolation of the gene encoding the human T-lymphocyte differentiation antigen Leu-2 (T8) by gene transfer and cDNA subtraction.

We report the isolation of genomic and cDNA clones encoding the human T-lymphocyte cell-surface differentiation antigen, Leu-2 (T8), by use of a combination of transfection, fluorescence-activated cell-sorting, and subtractive cDNA hybridization. We constructed a cDNA library with mRNA from a mouse L-cell transfectant in which the human Leu-2 gene is expressed and amplified. We identified Leu-2 cDNA clones by screening with a selected cDNA probe from a second amplified Leu-2 transfectant. This probe contained cDNA species not removed by hybridization with L-cell mRNA. A Leu-2 cDNA clone was used to isolate a genomic clone. Transfection with DNA from this clone resulted in a high number of Leu-2 transfectants. This approach can be used to clone genes coding for other cell-surface molecules.     

Human cardiac myosin heavy chain genes. Isolation of a genomic DNA clone and its characterization and of a second unique clone also present in the human genome

A gene sequence coding for myosin heavy chain (MHC) of human cardiac muscle was isolated by screening a human genomic library with a 32P-labelled 1.1kb SacI restriction fragment from a previously characterized cDNA clone specifying the light meromyosin and 3' untranslated region of mRNA encoding rabbit cardiac alpha-MHC. The DNA of this human genomic clone (lambda HCMHC8) hybridized much more strongly than did other clones isolated under similar, low stringency conditions both to the rabbit cDNA probe and to mRNA isolated from rat cardiac, but not skeletal, muscle tissue. Probe made from a DNA restriction fragment of lambda HCMHC8 hybridized a single 31S band of human ventricular mRNA. This size is identical to that of cardiac MHC mRNA of other species. Heteroduplex analysis showed hybridization of lambda HCMHC8 with exon segments in a rabbit cardiac MHC genomic clone (lambda MHC alpha 12/1). It also showed that lambda HCMHC8 spanned 14 kb of DNA and contained exon segments estimated to code for two-thirds of a MHC including the carboxylic acid terminus. By rescreening the library under more stringent conditions, where only DNA sequences having strong homology to cardiac MHC genes would be expected to hybridize, clones having restriction maps overlapping lambda HCMHC8 were isolated together with a unique clone (lambda HCMHC9). DNA gel blot hybridization of human genomic DNA with lambda HCMHC8 probe at medium stringency gave a pattern of restriction fragments similar to the restriction map of lambda HCMHC8. A weaker set of bands also appeared which corresponded in pattern to the map of lambda HCMHC9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type XII collagen: distinct extracellular matrix component discovered by cDNA cloning.

We have screened a cDNA library constructed from tendon fibroblast mRNA for the presence of collagenous coding sequences. Nucleotide sequence analysis of one isolated clone, pMG377, reveals that the clone encodes a polypeptide that is homologous to, yet distinctly different from, type IX short-chain collagen polypeptides. The structure of the conceptual translation product of the cDNA is also different from that of all other collagen types. Therefore, we have given the type IX-like collagen chain encoded by pMG377 the designation alpha 1(XII). Ribonuclease protection assays with single-stranded cRNA probes demonstrate that alpha 1(XII) mRNA is present in several tissues such as calvaria, tendon, and sternal cartilage of 17-day-old chicken embryo and in cornea from 6-day-old embryos. Using pMG377 as the hybridization probe, we isolated a fragment of the corresponding gene from a chicken genomic library. Partial nucleotide sequence analysis of the genomic clone DG12 shows that the exon/intron structure of the alpha 1(XII) collagen gene appears to be homologous to that of the alpha 1(IX) and alpha 2(IX) collagen genes. Our data demonstrate that types IX and XII collagen are two homologous members of a family of unique collagenous proteins that show tissue-specific patterns of expression. Based on their structure and the properties of their genes, we conclude that this family of collagens is distinctly different from that of fibrillar collagens.     

猪带绦虫抗原基因TS76的克隆及原核表达

目的　测定和分析自猪囊尾蚴表达型cDNA文库中筛选出的cDNA克隆 (TS76 )的序列 ,并进行该片段的原核表达研究。方法　采用PCR扩增重组 (噬菌体 (TS76中的TS76cDNA片段 ,亚克隆至 pUC18,得到的重组子用于测序 ,并对测定结果进行分析及同源性比较 ;将TS76片段亚克隆到原核表达载体pGEX - 1(T中 ,免疫印迹方法筛选得到能正确表达TS76片段的重组子 pGTS76 ;制备pGTS76原核表达产物 ,进行浓度梯度SDS -PAGE和Westernblotting分析。 结果　TS76克隆含 5 16对核苷酸 ,编码含 83个氨基酸残基的多肽 ,与EMBL数据库中的猪囊虫免疫原性蛋白质mRNA有 383bp的同源区域。重组质粒在大肠杆菌中表达的融合蛋白分子量为 34KD ,能与抗猪囊尾蚴兔血清产生很强的免疫反应。结论　分离到一个编码具较强免疫原性猪囊尾蚴抗原的基因。     

Expression of plasma glutathione peroxidase in human liver in addition to kidney, heart, lung, and breast in humans and rodents.

We analyzed the expression of plasma glutathione peroxidase (GSHPx-P) messenger RNA (mRNA) in mouse, rat, and human tissues, using a human GSHPx-P cDNA clone as the probe. Unlike the classical cellular glutathione peroxidase (GSHPx-1), GSHPx-P expression appears to be tissue-specific. In the mouse and rat, kidney expresses an mRNA at a high level detected with the human probe. A signal is also detected in mRNA isolated from mouse and rat heart, rat cardiac myocytes, mouse lung, epididymis, and the mammary gland of midpregnant mice. No signal is detected in mRNA isolated from mouse and rat liver, mouse brain, uterus, and testis. In human tissues, an mRNA hybridizing to GSHPx-P cDNA is present in liver, as well as kidney, heart, lung, breast, and placenta. We have shown that human kidney expresses a GSHPx-P mRNA, and not a GSHPx-P-like message, by isolating a cDNA clone from a human kidney library in lambda gt11. From the 412-nucleotide partial sequence of the kidney cDNA, which codes for the 40-170 amino acids of GSHPx-P including the TGA codon for selenocysteine, we found complete sequence identity of the kidney cDNA with GSHPx-P isolated from placenta. The expression of GSHPx-P mRNA in cell lines was also studied. There is some correlation of the expression of GSHPx-P in these cell lines with that in normal tissues. Cell lines that expressed GSHPx-P mRNA or protein included the human hepatocarcinoma HepG2, Hep3B cells, human kidney carcinoma A498 cells, and the human breast cancer SK-BR-3, T47D, MDA-MB-231, and AdrrMCF-7 cells. Cell lines that did not express GSHPx-P included human choriocarcinoma BeWo cells, human breast cancer MCF-7, ZR-75-1, and Hs578T cells, and mouse hepatoma Hepa-1 cells.