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Putative diazepam binding inhibitor peptide: cDNA clones from rat.   总被引:12,自引:10,他引:12       下载免费PDF全文
cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver. The clones contain an open reading frame corresponding to 87 amino acids. A signal sequence is not present. In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites. The size of the mRNA in all tissue examined is approximately 0.7 kilobase. Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes.  相似文献   

3.
Recent studies have suggested that many lysosomal enzymes, including cathepsin B (EC 3.4.22.1), may be synthesized as larger precursors and proteolytically processed to their mature forms. To determine the structure of the primary translation product of cathepsin B, we have screened a phage cDNA library for clones encoding rat liver cathepsin B. We synthesized two extended DNA oligonucleotides to use as hybridization probes: a 50-mer corresponding to the coding segment for residues 215-231 of mature cathepsin B and a 54-mer corresponding to residues 117-134. After screening 600,000 plaques, five clones were obtained that hybridized to the 32P-labeled 50-mer; of these, two (lambda rCB3 and lambda rCB5) also reacted with the 54-mer. DNA sequence analysis confirmed that lambda rCB3 and lambda rCB5 both encoded rat liver cathepsin B, and the translated sequence is in agreement with the sequence determined [Takio, K., Towatari, T., Katunuma, N., Teller, D. C. & Titani, K. (1983) Proc. Natl. Acad. Sci. USA 80, 3666-3670], except for a tryptophan for glycine substitution at residue 78 and the presence of two amino acids at the junction site of the light and heavy chains. Moreover, the DNA sequence reveals an open reading frame extending beyond the 5' (NH2 terminus), and the predicted COOH terminus of the coding sequence for the mature protein is extended by six amino acids. These results confirm that the biosynthesis of cathepsin B involves a larger precursor form and demonstrate the effectiveness of long oligonucleotide probes for screening to detect rare cloned mRNAs.  相似文献   

4.
The presence of the 29-amino acid peptide galanin and galanin-like immunoreactivity in the peripheral and central nervous system have been established. We describe below the construction of a 1.1-million-recombinant cDNA library derived from poly(A)+ RNA from pig adrenal medulla, rich in galanin-like immunoreactivity, and the isolation and characterization of clones encoding preprogalanin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to galanin-(7-19) and galanin-(22-29) were synthesized and used as hybridization probes to screen the cDNA library. Sequencing of two recombinant plasmids shows that pig galanin mRNA in the adrenal medulla encodes a precursor protein of 123 amino acids that is comprised of a leader sequence, a single galanin sequence, and a 59-amino acid sequence (galanin message-associated peptide) of as yet unknown function. The single galanin sequence, followed by a glycine (the amide donor), is flanked on both sides by pairs of lysine and arginine residues. RNA blot-hybridization analysis of poly(A)+ RNA from pig adrenal medulla reveals a single mRNA of approximately equal to 900-950 bases.  相似文献   

5.
cDNA clones coding for chicken cartilage link protein were isolated and sequenced. The DNA sequence for the entire core polypeptide of the mature link protein and the predicted signal peptide consists of 1065 nucleotides. The deduced primary translation product (355 amino acids) has a molecular mass of 40.7 kDa; the calculated molecular mass of the mature link protein core polypeptide (340 amino acids) is 39.06 kDa. The DNA sequence contains two tandemly arranged repeat sequences that may code for repeated functional domains of link protein involved in binding to hyaluronic acid. The mRNAs for chicken link protein are 6.0, 5.8, and 3.0 kilobase pairs, and the difference between the sizes of the RNA species lies in the 3' untranslated region.  相似文献   

6.
Several aldolase B clones from a human liver cDNA library have been identified by using a rabbit aldolase A cDNA as a hybridization probe. The most complete of these, pHL413, is 1389 base pairs long and covers approximately equal to 80% of the length of the mRNA, including 90% of the translated region. The cDNA, pHL413, was used to identify a genomic clone, lambda HG313, which encoded the remaining amino acids of human aldolase B. We demonstrate that the amino acid and nucleotide sequences of aldolase are strongly conserved even between different isozymes. Furthermore, in the 3'-untranslated regions of the mRNAs for the B isozyme of human and rat there is an extensive stretch of homology. Aldolase B lacks a cysteine at positions 72 and 338 and lacks a histidine at position 361. These residues, which are present in rabbit aldolase A, have previously been proposed to take part in catalysis. Our findings suggest that this may not be the case.  相似文献   

7.
cDNA clones encoding rodent IgE-binding factors (IgE-BF) were isolated from cDNA libraries of a rat-mouse T hybridoma that secretes IgE-suppressive factor (IgE-SF) upon incubation with rat IgE. COS7 cells transfected with two of the cDNAs expressed IgE-BF, which selectively potentiate an in vitro IgE response. IgE-BF expressed in COS7 cells are glycoproteins of approximately equal to 60 and approximately equal to 11 kDa. DNA sequence analysis of an IgE-BF cDNA revealed a 556-amino acid (62 kDa) protein coding region. The results suggest that IgE-potentiating and IgE-suppressive factors share common precursor polypeptides and that the 11-kDa IgE-BF is derived from a 60-kDa precursor.  相似文献   

8.
Isolation of cDNA clones encoding HLA-DR alpha chains.   总被引:6,自引:10,他引:6       下载免费PDF全文
HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.  相似文献   

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A cDNA library has been constructed from canine poly(A)+ mRNA. Clones containing cDNA inserts coding for prechymotrypsinogen 2 (isoelectric point = 7.1; Mr = 27,500), one of three canine pancreatic isoenzyme forms, were selected by colony hybridization using a cDNA probe synthesized from immunoselected prechymotrypsinogen 2 mRNA. To verify that cDNA clones code for prechymotrypsinogen 2 forms that translocate across rough endoplasmic reticulum membranes and fold into stable and identifiable secretory proteins, we conducted in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and optimal concentrations of glutathione and analyzed nascent translation products in their nonreduced state by two-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and fluorography. A near full-length chymotrypsinogen 2 cDNA and its primed extension were used to determine the nucleotide sequence for the entire coding region of prechymotrypsinogen 2 mRNA and 87 residues, including a poly(A) addition signal, in the 3' nontranslated region. The deduced amino acid sequence shows a 263-residue presecretory protein containing an 18-residue amino-terminal transport peptide (Met-Ala-Phe-Leu-Trp-Leu-Leu-Ser-Cys-Phe-Ala-Leu-Leu-Gly-Thr-Ala-Phe-Gly ), which we have previously shown to mediate the translocation of chymotrypsinogen 2 across the rough endoplasmic reticulum membrane. Following the transport peptide is a 245-residue proenzyme, which shows 82% and 80% sequence identity with bovine chymotrypsinogens A and B, respectively. Conserved among the three zymogens are 10 Cys residues that form five disulfide bonds in bovine chymotrypsinogens A and B and the residues that are required for zymogen activation, substrate binding, and catalytic activity.  相似文献   

11.
We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results of carboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.  相似文献   

12.
The primary structure of the precursor of urotensin I, a neuropeptide hormone from the caudal neurosecretory system of the carp Cyprinus carpio, has been determined by analyzing the nucleotide sequence of cloned DNA complementary to the mRNA encoding it. The precursor consists of 145 amino acid residues and the carboxyl terminus represents the 41-amino acid sequence of urotensin I, preceded by Lys-Arg and followed by Gly-Lys. Sequence homology as well as similar organization of the precursors of urotensin I and mammalian corticotropin-releasing factors suggest that they are evolutionarily related. RNA transfer blot analysis indicates that mRNA encoding the precursor of urotensin I is present only in the spinal cord and not in the brain, intestine, liver, or kidney of the carp.  相似文献   

13.
The primary structure of pig mitochondrial aspartate aminotransferase (mAspATase; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) precursor was deduced from its cDNA sequence. A library of cDNA clones was constructed from pig liver poly(A)+ RNA by applying the vector/primer method of Okayama and Berg [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170]. The library was screened for pig mAspATase sequences by using a mixture of eight oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known amino acid sequence of pig mAspATase residues 196-201. Two recombinant plasmids containing inserts of about 2500 and 2600 base pairs were selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the pig mAspATase precursor consists of the mature enzyme of 401 amino acid residues and an amino-terminal segment of 29 amino acid residues called the "presequence" that contains four basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The sequence of this 29-amino acid mAspATase precursor segment was compared with the presequences of other mitochondrial enzymes.  相似文献   

14.
Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.  相似文献   

15.
We have isolated three cDNA clones for beta 2-microglobulin, the small subunit of the major histocompatibility antigens. beta 2-Microglobulin makes up less than 0.1% of mouse liver protein, and its mRNA is approximately 0.03% of liver poly(A)+ mRNA. The cDNA clones were identified by screening 1400 cDNA clones made from 9--10S mouse liver poly(A)+ mRNA. The procedure for screening the cDNA clones involved binding pooled plasmid DNA to nitrocellulose filters and testing the ability of each filter to select beta 2-microglobulin mRNA. The filter-selected mRNAs were assayed for their ability to direct the synthesis of beta 2-microglobulin in translation reactions in vitro. The isolated clones were shown by nucleotide sequence analysis to encode beta 2-microglobulin. The positive-selection--hybridization assay has been modified to facilitate the screening of large numbers of cDNA clones, and the modified assay should allow the isolation of cDNAs corresponding to any mRNA whose in vitro translation products can be immunoprecipitated. These modifications are of particular value in the isolation of cDNA clones corresponding to rare species of mRNA.  相似文献   

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cDNA clones coding for a T-cell receptor beta-chain were isolated from a beef insulin/IAd-reactive T-cell hybridoma, A20.2.15, and its complete amino acid sequence was deduced. This beta-chain gene utilizes the same V beta segment as a thymocyte beta-chain gene (86T1) and rearranges to the 5' proximal J-C locus (J beta 1-C beta 1), thus providing definitive evidence of a beta-chain gene from a functional hybridoma that utilizes C beta 1. The amino acid sequence of the V beta gene in A20.2.15 is identical to 86T1, thus suggesting the absence of somatic mutation in the beta-chain of A20.2.15. Southern blot analysis revealed a somatic DNA rearrangement unique to the A20.2.15 hybridoma. The expression of this gene in the hybridoma was confirmed by RNA dot hybridization. All 24 beta-chain clones so far isolated from the A20.2.15 hybridoma contained C beta 1, suggesting that the beta-chain gene of the fusion partner BW5147 is not expressed in the hybridoma.  相似文献   

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We report here the isolation and nucleotide sequence of a complete cDNA clone encoding a photosystem I (PS I) polypeptide that is recognized by a monoclonal antibody made against photosystem II (PS II) chlorophyll a/b-binding (CAB) proteins. The deduced sequence of this PS I protein shows 30% overall identity to PS II CAB sequences, and two long segments within this protein show 50% and 65% identity to the corresponding segments in the PS II CAB polypeptides. Even though the sequence of this PS I CAB protein is substantially divergent from PS II CAB sequences, their hydropathy plots are very similar and suggest they all traverse the thylakoid membrane three times. A segment of the PS I CAB polypeptide shows similarity to the functionally analogous beta subunits of the antenna proteins of purple bacteria. In contrast, no homology was observed between these bacterial proteins and PS II CAB polypeptides.  相似文献   

20.
Mixed oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identity with rabbit skeletal muscle phosphorylase kinase gamma-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.  相似文献   

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