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1.
T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116-120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Calpha2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells.  相似文献   

2.
Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.  相似文献   

3.
4.
Many endogenous and xenobiotic chemicals are metabolized to epoxides which may be enzymatically hydrated, via microsomal epoxide hydrolase (mEH), to less reactive dihydrodiol derivatives. On the basis of the reported rabbit mEH amino acid sequence [F. S. Heinemann and J. Ozols (1984) J. Biol. Chem. 259, 797-804], we constructed a 35 base oligonucleotide which was used to screen rabbit liver cDNA libraries. Overlapping rabbit mEH clones were isolated and the full-length cDNA sequence of 1653 bp was determined. The rabbit nucleotide sequence has a high degree of similarity (greater than 75%) with cDNA sequences reported for rat and human mEH. Northern blot analyses with fragments of the rabbit cDNA demonstrate that mEH messenger RNA (mRNA) is expressed constitutively in the liver and induced following exposure to phenobarbital or polychlorinated biphenyls. Constitutive expression of mEH mRNA is also observed in rabbit kidney, testes, and lung. Using benzo[alpha]pyrene-4,5-oxide as substrate, mEH enzymatic activity is shown to correlate closely with tissue levels of mEH mRNA. Southern blot analyses of rabbit DNA suggest that the mEH gene exists as a single copy per haploid genome. The mEH amino acid sequences of the human and rat were compared to that of the deduced rabbit protein in order to analyze the degree of conservation and hydropathy profiles in these species. This comparison permitted the formulation of a computer-assisted model of mammalian mEH as it may relate to the microsomal membrane.  相似文献   

5.
We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third cDNA is about 72-74% homologous to the other two. All three are 55-60% homologous to rat liver cytochrome P-450b cDNA. The amino acid sequences derived from the cDNA sequences are about 50% homologous to those of rat liver cytochrome P-450b and rabbit liver cytochrome P-450 (form 2). The degree of homology differs substantially in different regions of the protein. The hydrophobicity profiles of these five mammalian cytochromes P-450 are very similar and contain up to eight regions of hydrophobicity that are long enough to span a membrane. These results indicate that these three cDNAs code for rabbit liver cytochromes P-450 which are different from any rabbit liver cytochrome P-450 for which amino acid sequence information is published. These cDNAs are part of a family of genes that are related to rabbit liver cytochrome P-450 (form 2) and rat liver cytochrome P-450b which are the major phenobarbital-inducible forms. The divergence of amino acid sequence between the rat and rabbit forms and the divergence of nucleotide sequences of silent sites in the two most closely related rabbit forms suggest that cytochromes P-450 have a relatively high rate of amino acid divergence compared to many other vertebrate proteins.  相似文献   

6.
Cloning and sequencing of a human liver carboxylesterase isoenzyme   总被引:1,自引:0,他引:1  
A human liver lambda gt11 library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxylesterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.  相似文献   

7.
Characterization of a cloned cDNA encoding rabbit myelin P2 protein   总被引:2,自引:0,他引:2  
Myelin P2 is a 14,800-Da cytosolic protein found in rabbit sciatic nerves. It belongs to a family of fatty acid binding proteins and shows a 72% amino acid sequence similarity to aP2/422, the adipocyte lipid binding protein, a 58% sequence similarity to rat heart fatty acid binding protein, and a 40% sequence similarity to cellular retinoic acid binding protein. In order to isolate cDNA clones representing P2, a cDNA library was constructed from poly(A+) RNA isolated from sciatic nerves of 10-day-old rabbit pups. By use of a mixed synthetic oligonucleotide probe based on the rabbit P2 amino sequence, 12 cDNA clones were selected from about 25,000 recombinants. Four of these were further characterized. They contained an open reading frame, which when translated, agreed at 128 out of 131 residues with the known rabbit P2 amino acid sequence. These cDNAs recognize a 1.9-kilobase mRNA present in sciatic nerve, spinal cord, and brain, but not present in liver or heart. The levels of P2 mRNA parallel myelin formation in sciatic nerve and spinal cord with maximal amounts being detected at about 15 postnatal days. This initial study will allow characterization of the P2 gene and its regulation, as well as further studies into the role of P2, the first metabolically active myelin-specific protein to be characterized at the genetic level.  相似文献   

8.
Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme [Costa et al. (1990) Toxicol. Appl. Pharmacol. 103, 66-76]. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined by (1) gas-phase sequencing of the intact protein and (2) peptide fragments from lysine and arginine digests. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. A clone was isolated and sequenced, and contained a 1294-bp insert encoding an open reading frame of 359 amino acids. Northern blot hybridization with RNA isolated from various rabbit tissues indicated that paraoxonase mRNA is synthesized predominately, if not exclusively, in the liver. Southern blot experiments suggested that rabbit paraoxonase is coded by a single gene and is not a family member of closely related genes. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Each of the human clones appeared to be polyadenylated at a different site, consistent with the absence of the canonical polyadenylation signal sequence. Of potential significance with respect to the paraoxonase polymorphism, the derived amino acid sequence from one of the partial human cDNA clones differed at two positions from the full-length clone. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue [Furlong et al. (1991) Biochemistry (preceding paper in this issue)]. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.  相似文献   

9.
Two recombinant clones, pA2 and pA3, containing cDNA sequences for human aldolase B have been isolated from a full length human liver cDNA library. The larger one, pA3, has been subcloned in M13 phage and completely sequenced with the chain terminator method. The sequence covers 1,600 nucleotides including the whole coding region (1,050 nucleotides), 67 nucleotides from the 5' non-coding region and the whole 3' non-coding region, 440 nucleotides long, down to the poly-A tail. Comparison with rabbit aldolase A and with a partial sequence of rat aldolase B, shows a homology of about 76% for aldolase A and of about 94% for aldolase B, which indicates that the sequenced cDNA codes for the liver isoenzyme. This is the first complete sequence reported for human aldolase B. The pA3 clone strongly hybridizes to 18S mRNA from human adult liver as expected from the size of the isolated cDNA.  相似文献   

10.
K Joh  T Mukai  H Yatsuki  K Hori 《Gene》1985,39(1):17-24
The nucleotide sequence of aldolase A mRNA in rat skeletal muscle was determined using recombinant cDNA clones and a cDNA synthesized by primer extension. The sequence is composed of 1343 nucleotides (nt) except for the poly(A) tail. Based on the sequence analysis we have deduced an open reading frame with 363 amino acids (aa) (Mr 39134). The sequence suggests several nt polymorphisms in the mRNA population, one of which causes an aa change. The determined sequence of rat aldolase A mRNA was compared with the published ones of rabbit aldolase A or rat aldolase B mRNAs. The homology between rat and rabbit aldolase A mRNA sequences is greater than that between rat aldolase A and B mRNA sequences. Multiple aldolase A mRNAs having different Mrs were detected in the various tissues, and appeared to be expressed in a tissue-specific manner. Further analysis suggests that differences in mRNA length are due to differences in the 5'-noncoding terminal region.  相似文献   

11.
Classical phenylketonuria, an inborn error in metabolism, is caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. The identification of putative cDNA clones coding for rat liver phenylalanine hydroxylase by hybrid-selected translation has previously been reported [Robson, K. J., Chandra, T., MacGillivray, R. T. A., & Woo, S. L. C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4701-4705]. The authenticity of the clones, however, could not be definitively ascertained at the time because of a lack of amino acid sequence data of the enzyme in the literature. Purified rat liver phenylalanine hydroxylase was subjected to cyanogen bromide treatment, and the resulting fragments were used for N-terminal amino acid sequence analysis. The partial amino acid sequence was then compared to that deduced from an open reading frame in the nucleotide sequence of the cDNA clones. A perfect match of 17 amino acid residues was found between the two sequences following a unique methionine codon present in the nucleotide sequence, thereby providing unambiguous evidence for the identity of the rat liver phenylalanine hydroxylase cDNA clones.  相似文献   

12.
cDNA clones encoding rat liver mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, were isolated from a rat liver cDNA library carried in lambda gt 11, by screening with affinity purified polyclonal rabbit anti-rat liver MBP antibodies. The nucleotide sequence of the cDNA determined by the dideoxy method revealed the complete amino acid sequence of the MBP (226 residues). The NH2-terminal residue of the MBP, glutamic acid, was preceded by a predominantly hydrophobic stretch of 18 amino acids, which was assumed to be a signal peptide. Near the NH2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence Gly-X-Y. Here, X and Y were frequently proline and lysine. Three proline and lysine residues were hydroxylated, and one of the latter appeared to link to galactose. Computer analysis of several lectins for sequence homology suggested that the COOH-terminal quarter of the MBP is associated with the calcium binding as well as carbohydrate recognition.  相似文献   

13.
Cloning and regulation of rat apolipoprotein B mRNA   总被引:3,自引:0,他引:3  
Recombinant cDNA clones that code for apolipoprotein B(apoB) were isolated from a rat liver cDNA library, using synthetic oligonucleotide probe derived from the sequence of human apoB cDNA. The nucleotide and deduced amino acid sequences of the rat apoB clone pRB5, 1.2 kb in length, showed 83% and 84% homology to those of human apoB. Northern blot analysis revealed that rat apoB cDNA probe cross-reacts with human and rabbit apoB mRNA sequences and the size of those mRNAs, approximately 15 kb long, were not discernibly different. In addition, apoB mRNA was abundant only in the liver and intestine. Finally, cholesterol feeding to rats for six weeks resulted in a several-fold increase in the level of apoB mRNA in the liver.  相似文献   

14.
用基因组DNA剪接技术克隆SIgA相关基因   总被引:1,自引:0,他引:1  
目的:克隆分泌型IgA(SIgA)相关基因--J链基因(IgJ)、多聚免疫球蛋白受体基因(pIgR)和IgA重链恒定区基因(IGHA),为进一步构建SIgA真核表达质粒奠定基础。方法:采用本室建立的"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果:PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明本实验获得的上述基因与GenBank中的目标基因序列完全一致。结论:本文通过基因组DNA剪接技术成功克隆人类SIgA三个相关基因,提示此技术是合成多外显子cDNA的有效手段。  相似文献   

15.
We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.  相似文献   

16.
We have isolated one full-length cDNA clone, termed pHP1, and a number of clones of shorter insert lengths, tentatively called b14, b46, etc., all encoding phenobarbital- (PB-) inducible forms of rabbit liver microsomal cytochrome P-450, and determined their nucleotide sequences. The polypeptides encoded by these cDNAs can be classified into five types, represented by HP1, b14, b46, b52, and b54, the deduced amino acid sequences of which are more than 95% similar to one another. Amino acid differences among them total 24 positions, which are distributed over the entire sequence, in contrast to the microheterogeneity observed in two PB-inducible rat liver microsomal cytochromes P-450 (P-450b and P-450e). The primary structure deduced for the HP1 protein is 97% similar to that determined for rabbit P-450 LM2 (form 2), which has been purified by Coon and co-workers [van der Hoeven, T. A., Haugen, D. A., & Coon, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 569-675; Haugen, D. A., & Coon, M. J. (1976) J. Biol. Chem. 251, 7929-7939] as the major PB-inducible form of rabbit liver microsomal cytochrome P-450. The amino acid sequence of P-450(1), which we have purified as the major PB-inducible rabbit liver cytochrome P-450, was partially determined with the sequence reported for P-450 LM2 as a reference. The two sequences are closely similar to each other, but at least two amino acid differences can be detected between them.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
Isolation of a cDNA encoding the rat liver S-adenosylmethionine synthetase   总被引:4,自引:0,他引:4  
We have isolated cDNA clones encoding the rat liver S-adenosylmethionine synthetase by means of immunological screening from a phage lambda gt 11 expression library containing cDNA synthesized from adult rat liver poly(A)-RNA. The amino acid sequence deduced from the cDNA indicates that the rat liver enzyme for this protein contains 397 amino acid residues and has a molecular mass of 43697 Da. The deduced amino acid sequence of rat liver S-adenosylmethionine synthetase was 68% similar to those of yeast S-adenosylmethionine synthetases encoded by two unlinked genes SAM1 and SAM2. The rat liver S-adenosylmethionine synthetase also shows 52% similarity with the deduced amino acid sequence of the MetK gene encoding the S-adenosylmethionine synthetase in Escherichia coli.  相似文献   

18.
A 1.7-kilobase pair cDNA clone encoding 3-hydroxyisobutyrate dehydrogenase has been isolated by screening a rat liver lambda gt11 library with a 17-base oligonucleotide probe which corresponds to a portion of the N-terminal amino acid sequence of rabbit liver 3-hydroxyisobutyrate dehydrogenase. The cDNA contains an open reading frame of 1038 base pairs which includes an amino acid sequence that matches the N-terminal 35 amino acid sequence of rabbit 3-hydroxyisobutyrate dehydrogenase at 33 residues. The cDNA predicts a 300-amino acid mature protein with an amino acid composition and molecular weight very similar to that of rabbit liver 3-hydroxyisobutyrate dehydrogenase. Northern blot analysis of total RNA from several rat tissues shows an mRNA of approximately 2.0 kilobase pairs in each tissue. Relative mRNA levels were: kidney greater than liver = heart greater than muscle. The amino acid sequence of 3-hydroxyisobutyrate dehydrogenase shows similarity to several other pyridine nucleotide-dependent dehydrogenases. The resemblance to malate and lactate dehydrogenases suggests that the nucleotide-binding domain is located in the N-terminal region of the protein.  相似文献   

19.
A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.  相似文献   

20.
In a study of the regulation of gene expression in liver cells, a strain of dedifferentiated cells (C2) derived from the rat hepatoma line H4IIEC3 was transfected with DNA from human liver. After growth of these C2 variants in a glucose-free medium, revertants were selected which were characterized by the expression of a complete set of liver functions. A 4.3 kb human DNA sequence was detected with an Alu sequence probe in cells of four independent revertant clones and was shown to be an extrachromosomal, covalently closed duplex DNA. This molecule, called HALF1 for reversion inducing sequence, was cloned and transfected into C2 cells. Analyses of the transfectants indicated a correlation between the introduction of this cloned genomic human DNA sequence and the recovery of hepatic traits.  相似文献   

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