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1.
We have isolated the rabbit immunoglobulin b9 Ck light chain gene and compared its nucleotide sequence with the b4, b4var , b5 and bas Ck sequences. In spite of the high number of substitutions found between the different rabbit Ck coding regions, only very few changes are silent. Furthermore, the nucleotide changes are clustered in segments which correlate with the bends and helical regions found in the tertiary structure of the Ck domain of the protein. The flanking regions present a higher degree of conservation than the coding regions. The two genomic EcoRI fragments hybridizing to a b4cDNA probe have been correlated with the two distinct loci, Ck1 and Ck2 : one encodes for the nominal b9 Ck allotype and the other contains the information for the bas Ck region. The b allotypes are true alleles which could have evolved by intergenic conversion.  相似文献   

2.
We report the sequence of a cDNA clone encoding the entire variable and constant regions of a rabbit kappa light chain of b5 allotype. The deduced amino acid sequence of the variable region (positions 1-95) is 86% homologous to that of a b4 light chain protein [BS-1) (1) but the b4 and b5 constant regions are only 74% homologous. Comparison of this DNA sequence to that of a cDNA clone encoding a b4 constant region shows that the kappa allotypes b4 and b5 have diverged significantly more in their coding region than in the 3' untranslated regions (86% vs 96% nucleotide sequence homologies). This implies either a function for the 3' untranslated region with evolutionary pressures to conserve or an accelerated divergence of the coding regions.  相似文献   

3.
Rabbit Ig kappa 1b6 gene structure   总被引:1,自引:0,他引:1  
Previous studies employing Southern blot analyses have detected multiple kappa-homologous sequences within EcoRI-digested DNA isolated from kappa 1b6 homozygous rabbits and kappa 1b6 L chain secreting RMH H158 cell line. These results are very unexpected because the published partial protein sequence for the kappa 1b6 C region is incompatible with an EcoRI restriction endonuclease recognition sequence at the nucleotide level for this allotype. To determine their identity, the kappa-homologous sequences were isolated from DNA extracted from a kappa 1b6 L chain secreting RMH H158 cell line by molecular cloning. Structural analyses demonstrated these sequences to contain genetic information encoding the majority of the kappa 1b6 L chain gene locus. The protein sequence deduced from the kappa 1b6 C region gene was shown to differ from the published partial kappa 1b6 C region protein sequence at five amino acid positions. One of these differences results in a glycine to serine interchange that introduces an EcoRI restriction endonuclease recognition site within the kappa 1b6 C region gene. Subsequent genomic Southern blot analyses confirmed this structural assignment. Based on these data, the EcoRI-sensitive kappa-homologous fragments present within the genomes of the RMH H158 cell line and kappa 1b6 homozygous rabbits represent the nominal kappa 1 gene and not an alternative kappa isotype or kappa pseudogene. Rabbit Ig kappa 1 allelic nucleotide sequence homology comparisons have shown the isolated kappa 1b6 J-C gene locus to display common structural features previously identified in other kappa 1 alleles.  相似文献   

4.
Our previous analysis of an immunoglobulin gene encoding the rabbit kappa chain of b4 allotype revealed that of the five J-like sequences in the J kappa cluster of this gene, only one, J2, appeared to be functional. This unusual ratio of J pseudogenes to functional J genes is unique among all J clusters of light and heavy chain genes of all species examined to date, including the cluster from the rabbit kappa 2 isotype, and must have consequences for diversity generation of b4 immunoglobulins. The fact that the only two known b5 J kappa sequences are different from the functional J2 of the b4 allotype prompted investigation of the b5 J kappa cluster to determine whether it resembled the b4 cluster, or the more typical mouse or human J kappa clusters. Our analysis of the b5 gene reveals a J kappa cluster strikingly similar to that of b4; apparent defects occur in all J sequences except J2. Although J2 is apparently functional, it differs from the J2 of the b4 locus by four nucleotide and three amino acid substitutions. The unusually high degree of sequence similarity previously observed between the b4 and b5 loci in the noncoding (vs the coding) regions extends through the newly sequenced DNA segment and remains an enigma.  相似文献   

5.
Comparison of latent and nominal rabbit Ig VHa1 allotype cDNA sequences   总被引:1,自引:0,他引:1  
The genetic basis for the expression of a latent VH allotype in the rabbit was investigated. VH region cDNA libraries were produced from spleen mRNA derived from a homozygous a2a2 rabbit expressing an induced latent VHa1 allotype and, for comparison, from a normal homozygus a1a1 rabbit expressing nominal VHa1 allotype. The deduced amino acid sequences of the nominal VHa1 cDNA were concordant with previously published VHa1 protein sequences. A comparison of two complete VH-DH-JH and six partial VHa1 sequences reveals highly conserved sequence within VH framework regions (FR) and considerable diversity in complementarity-determining regions and D region sequences. Two functional JH genes or alleles are evident. Amino acid sequencing of the N-terminal 15 residues of pooled affinity-purified latent VHa1 H chain showed complete sequence identity with the nominal VHa1 sequences. Possible latent VHa1-encoding cDNA clones, derived from the a2a2 rabbit, were selected by hybridization with oligonucleotide probes corresponding to the VHa1 allotype-associated segments of the first and third framework regions (FR1 and FR3). cDNA sequence analysis reveals that the 5' untranslated regions of nominal and latent VHa1 cDNA were virtually identical to each other and to previously reported sequences associated with VHa2 and VHa-negative genes. Moreover, some latent VHa1 genes encode FR1 segments that are essentially homologous to the corresponding segment of a nominal VHa1 allotype. In contrast, other putative latent genes display blocks of VHa1 sequence in either FR1 or FR3 that are flanked by blocks of sequence identical to other rabbit VH genes (i.e., VHa2 or VHa-negative). These composite sequences may be directly encoded by composite germ-line VH genes or may be the products of somatically generated recombination or gene conversion between genes encoding latent and nominal allotypes. The data do not support the hypothesis that latent genes are the result of extensive modification by somatic point mutation.  相似文献   

6.
We report the sequence of a cDNA encoding a rabbit immunoglobulin gamma heavy chain of d12 and e14 allotypes with high homology to partial cDNA sequences from rabbits of d11 and e15 allotypes. The encoded rabbit protein shows homologies with human (68-70%) and mouse (60-63%) gamma chains. The nucleotide sequence homologies of the CH domains range from 76-84% with human and 64-76% with mouse sequences. Comparison of the portion of VH encoding amino acid positions 34-112 with a previously determined VH sequence of the same allotype shows high conservation of sequences in the second and third framework segments but more marked differences both in length and encoded amino acids of the second and third complementarity-determining regions (CDRs). We also found a high degree of homology with a human genomic V-region, VH26 (77%) and a remarkable similarity between rabbit and human second CDR sequences and human genomic D minigenes. These results provide additional evidence that D minigene sequences share information with the CDR2 portion of VH regions.  相似文献   

7.
Molecular cloning of rabbit gamma heavy chain mRNA.   总被引:5,自引:1,他引:4       下载免费PDF全文
A cDNA library of rabbit spleen mRNA was screened for immunoglobulin heavy chain sequences. In this paper we report the nucleotide sequence of two cDNA clones containing part of the constant region of the rabbit gamma heavy chain mRNA. The sequence encodes part of the CH2 domain (amino acids 268 to 340), the entire CH3 domain (amino acids 341 to 447) and the 3' untranslated region. This nucleotide sequence has been compared to the corresponding sequences of mouse gamma 1, gamma 2a and gamma 2b genes. The homologies between rabbit gamma chain gene sequence and each of the mouse gamma chain gene sequences are of the same magnitude order. This comparison shows that the CH2 domains are more homologous to each other than CH3 domains or 3' untranslated sequences. The presence of species specific nucleotide positions suggests that mouse gamma chain genes could have evolved from a common ancestor shortly after the mouse-rabbit species separation. Genomic blot analysis of rabbit liver DNA with the rabbit C gamma probes shows a limited number of related sequences, with little restriction site polymorphism between individual rabbits.  相似文献   

8.
Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.  相似文献   

9.
The rabbit genomic segments for the soluble cytochrome b5 (b5) and microsomal b5 were amplified and isolated, respectively, by means of the polymerase chain reaction using primers corresponding to various portions of the open reading frame of microsomal b5 cDNA. The DNA sequence analysis revealed that the soluble b5 gene has an extra 24 nucleotide long insert which encodes a C-terminal amino acid and a termination codon which are specific to the soluble b5. Except for the insert, the sequences of the soluble and microsomal b5 genes are identical with each other from the 5' end to the 3' end of the open reading frame of the microsomal b5 cDNA. Comparison of the genomic sequences with the cDNA sequences suggested that the soluble and microsomal genes are intronless within their open reading frames. These data indicate that rabbit soluble and microsomal b5 mRNAs are encoded by two highly conserved but separate genes.  相似文献   

10.
Synthetic DNA oligomers specific for the VHa allotypes of rabbit Ig genes have been used to identify latent allotypic sequences in homozygous a1 and a2 rabbits. Two Ig VH pseudogenes containing latent a3 regions have been cloned from the genome of a homozygous a2 rabbit. Analysis of the regions associated with allotype expression indicates that these two pseudogenes contain VHa- sequences in framework region 1 (FR1) and VHa3 sequences in FR3. One gene has undergone an unusual rearrangement with a third VH gene, deleting their intervening sequences and recombining in FR3 with sequences 5' to the leader exon. Our results demonstrate the presence of latent VH sequences in the genomic DNA of normal rabbits and suggest that a mechanism such as gene conversion is responsible for expression of genetically-unexpected Ig VH genes.  相似文献   

11.
A cDNA plasmid insert encoding the constant (C) region of a rabbit immunoglobulin-lambda light chain was used as a probe for screening a rabbit liver genomic DNA cosmid library. This allowed the isolation and identification of four distinct C lambda genes, designated C lambda 1, C lambda 2, C lambda 3, and C lambda 4, which were shown to be widely separated from each other along chromosomal DNA. Their nucleotide sequences have been determined. No in-frame termination codons were found within the coding regions. The C lambda 1, C lambda 2, and C lambda 3 sequences are quite similar to each other, but share less homology with the C lambda 4 gene or the cDNA-C lambda sequence used as a probe. The C lambda gene coding for the cDNA sequence was not isolated. Translation of the C lambda 1, C lambda 2, and C lambda 3 sequences predicts a Cys-Pro carboxy-terminal amino acid sequence, as found so far only for horse lambda-chains. Compared to the other rabbit C lambda genes, the C lambda 3 sequence exhibits two deletions, one of 9 bp, the other of 3 bp. The latter occurs at the same position as in the mouse C lambda 2 and C lambda 3 genes. These two deletions are located in the loops between anti-parallel beta-pleated sheets of the C lambda domain. When the C lambda nucleotide sequences from man, mouse, and rabbit are compared, there is less divergence within the same species than for interspecies comparisons. Possible genetic implications of this finding are discussed.  相似文献   

12.
A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.  相似文献   

13.
The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).  相似文献   

14.
A sequence of 10,621 base-pairs from the alpha-like globin gene cluster of rabbit has been determined. It includes the sequence of gene zeta 1 (a pseudogene for the rabbit embryonic zeta-globin), the functional rabbit alpha-globin gene, and the theta 1 pseudogene, along with the sequences of eight C repeats (short interspersed repeats in rabbit) and a J sequence implicated in recombination. The region is quite G + C-rich (62%) and contains two CpG islands. As expected for a very G + C-rich region, it has an abundance of open reading frames, but few of the long open reading frames are associated with the coding regions of genes. Alignments between the sequences of the rabbit and human alpha-like globin gene clusters reveal matches primarily in the immediate vicinity of genes and CpG islands, while the intergenic regions of these gene clusters have many fewer matches than are seen between the beta-like globin gene clusters of these two species. Furthermore, the non-coding sequences in this portion of the rabbit alpha-like globin gene cluster are shorter than in human, indicating a strong tendency either for sequence contraction in the rabbit gene cluster or for expansion in the human gene cluster. Thus, the intergenic regions of the alpha-like globin gene clusters have evolved in a relatively fast mode since the mammalian radiation, but not exclusively by nucleotide substitution. Despite this rapid mode of evolution, some strong matches are found 5' to the start sites of the human and rabbit alpha genes, perhaps indicating conservation of a regulatory element. The rabbit J sequence is over 1000 base-pairs long; it contains a C repeat at its 5' end and an internal region of homology to the 3'-untranslated region of the alpha-globin gene. Part of the rabbit J sequence matches with sequences within the X homology block in human. Both of these regions have been implicated as hot-spots for recombination, hence the matching sequences are good candidates for such a function. All the interspersed repeats within both gene clusters are retroposon SINEs that appear to have inserted independently in the rabbit and human lineages.  相似文献   

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18.
Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.  相似文献   

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20.
K Sakai  M Hirai  J Kudoh  S Minoshima  N Shimizu 《Genomics》1992,14(1):175-178
We have isolated and sequenced complementary DNA (cDNA) for the human 80K-L protein, a major substrate for protein kinase C and the human homologue of an 80- to 87-kDa bovine protein named MARCKS (myristoylated alanine-rich C kinase substrate). The human 80K-L cDNA encodes a protein of 332 amino acids with a calculated molecular weight of 31,534. Homology comparisons of the nucleotide sequences of the cDNAs indicated that their 3'-untranslated regions are more homologous than the coding regions. Spot blot hybridization using flow-sorted human chromosomes indicated that the gene encoding the 80K-L protein, designated MACS, maps to the q15----qter region of human chromosome 6, and it also suggested that a genomic region with a sequence homologous to the 3'-untranslated region of the 80K-L mRNA exists on chromosome 21.  相似文献   

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