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1.
Recombinant cDNA clones corresponding to the human 1.9kb HindIII repetitive element have been isolated from a cDNA library of liver cytoplasmic polyadenylated RNA. These cDNAs share 95% homology with the reported genomic DNA sequence and a similar amount of homology at the amino acid level with putative coding sequences (see preceding article by Mottez et al). They were isolated as two of four false positives from a human cDNA library in lambda gt11 and were selected with an antibody to an unrelated enzyme. These results provide direct evidence that this repetitive element is transcribed to form poly(A)+ RNA which could be translatable. Also, these observations may add to our understanding of the sources of false positives which are frequently observed in screens of cDNA libraries with antibodies as probes.  相似文献   

2.
Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.  相似文献   

3.
Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.  相似文献   

4.
Y Haraguchi  T Uchino  M Takiguchi  F Endo  M Mori  I Matsuda 《Gene》1991,107(2):335-340
Carbamyl phosphate synthetase I (CPSI) is the first enzyme involved in urea synthesis. CPSI deficiency is an autosomal recessive disorder characterized by hyperammonemic coma in the neonatal period. To analyze the enzyme and gene structures, and to elucidate the nature of mutations in CPSI deficiency, we isolated cDNA clones encoding human liver CPSI. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using 5', middle, and 3' fragments of the rat CPSI cDNA as probes. Seven positive clones covered the full-length cDNA sequence with an open reading frame encoding a precursor polypeptide of 1500 amino acids (aa) (deduced Mr, 164,828) with a putative N-terminal presequence of 38 or 39 aa, a 5'-untranslated sequence of 118 bp and a 3'-untranslated sequence of 597 bp. Comparison with the rat CPSI cDNA showed that the deduced aa sequence of the human liver CPSI precursor is 94.4% identical to the rat enzyme precursor. A molecular analysis was made of the genomic DNA from three patients with CPSI deficiency. Heterogeneity of hybridized fragments that may or may not be the cause of the deficiency was apparent on the DNA blots from tissues from one patient.  相似文献   

5.
A lambda gt11 cDNA library was constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats and screened with a human tropoelastin cDNA clone. DNA sequence analysis of several overlapping rat clones confirmed the presence of DNA sequences coding for murine tropoelastin and DNA sequences coding for the 3'-untranslated region of the rat tropoelastin mRNA. Northern blot analysis of total RNA from aortic tissue of neonatal rats using oligonucleotide probes derived from these rat tropoelastin cDNAs demonstrated the presence of a 3.5-kilobase tropoelastin mRNA. The size of this rat tropoelastin mRNA agrees with previous reports for the size of the mRNA coding for tropoelastin in tissue from several vertebrate species but contrasts with several reports suggesting the presence of a higher molecular weight mRNA species responsible for the synthesis of tropoelastin in rodent tissue.  相似文献   

6.
E F Sato  Y Tanaka  K Utsumi 《FEBS letters》1989,244(1):108-112
cDNA clones of guinea pig neutrophil 33 kDa protein, a lipocortin like-protein, were isolated from two lambda gt10 libraries and one primer-extended lambda gt10 library of guinea pig neutrophils using synthetic oligonucleotide probes or cDNA fragment probe. The cDNA consists of 1389 nucleotides, and contains 1038 nucleotides encoding 346 amino acids of 33 kDa protein and a poly(A) tail. The deduced amino acid sequence shows high homology with those of lipocortin 1 from human U937 cells (89% homology) and rat lung (86%).  相似文献   

7.
A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).  相似文献   

8.
9.
3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.  相似文献   

10.
Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.  相似文献   

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