Molecular cloning of rabbit gamma heavy chain mRNA.

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.     

The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase

Recently we reported the sequence of a cDNA clone (pUOX-1), isolated from a lambda gt11 cDNA library, which encoded for rat liver urate oxidase (EC 1.7.3.3), but this clone lacked the nucleotide sequences encoding the N-terminal region for this enzyme. Using the cDNA insert from the pUOX-1 clone as a probe, we have now isolated a full length cDNA clone, pUOX-2, from a lambda gt10 library by plaque hybridization. Nucleotide sequence analysis of the pUOX-2 clone showed that it has 1379 base pairs with an open reading frame coding for 303 amino acid residues corresponding to a molecular mass of 34,931 daltons. In addition to the open reading frame the pUOX-2 contains 439 bp of 3'-untranslated and 41 bp of 5'-untranslated sequences. The consensus polyadenylation signal AATAAA precedes a stretch of poly(A)+ residues at the 3' end.     

Isolation, sequence analysis and characterization of cDNA clones coding for the C chain of mouse C1q. Sequence similarity of complement subcomponent C1q, collagen type VIII and type X and precerebellin.

A mouse macrophage lambda gt11 cDNA library was screened using a genomic DNA clone coding for the C-chain gene of human C1q. Approximately 600,000 recombinant phage plaques were hybridized with peroxidase-labeled human C-chain probe and detected by enhanced chemiluminescence. Five positive clones were obtained. The size of the full-length cDNA is 1019 bp. The sequence identity of the nucleotide sequence with human C1q C chain is 79%, the identity of the deduced amino acid sequences is 73%. The mouse C1q C chain exhibits the same structural features as the human C chain, e.g. conservation of the cysteine residues. Like the mouse A chain, the mouse C chain has an RGD sequence that may be recognized by receptors of the integrin family. No RGD sequences have been found in any of the human C1q chains. The size of the C-chain mRNA (1.2 kb) and its tissue distribution (macrophages being the cell type with the highest mRNA concentration) are identical to the mRNA of the mouse A and B chains. Alignment of human and mouse C1q A, B and C chains exhibits two blocks of highly conserved residues within the C-terminal globular regions. Three other proteins, collagen type VIII and type X and precerebellin share this similarity with C1q, indicating the structural and probably functional importance of these regions within the non-collagenous domains of the molecules.     

Cloning and sequencing of immunoglobin lambda-chain cDNA in american mink (Mustela vison

cDNA library in the lambda gt11 phage was constructed using poly (A+)-mRNA from mink spleen as a template. Immunoscreening of the library allowed the identification of 2 lambda-related clones containing 370 and 803 bp insertion (lambda IGL-1 and lambda IGL-2). Analysis of the primary structure of lambda IGL-2 demonstrated that it contains a large portion of V lambda-segment, J lambda-segment, C lambda-gene and its 3'-untranslated part. The nucleotide sequences known for the immunoglobulin genes were compared to the sequence of the lambda IGL-2 clone. The highest degree of homology was established for the rabbit lambda-genes, this being 63, 94 and 72% for the RF3 region of V lambda-segment, J lambda-segment and C lambda-region, respectively.     

cDNA clones encoding rabbit immunoglobulin lambda chains. Evidence for length variation of the third hypervariable region and for a novel constant region.

Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

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.     

Isolation and sequence of sheep Ig H and L chain cDNA

Sheep lymphocyte poly(A+) RNA was used as a template for the enzymatic synthesis of cDNA before cloning into the expression vector lambda gt11. Screening of the cDNA library with mAb probes resulted in the isolation of two recombinant phages containing Ig coding sequences of 704 bp and 925 bp. These were inserted into the EcoRI site of pUC18 and named pSLC (sheep Ig L chain) and pSHC (sheep Ig H chain). The insert in pSLC revealed sequence homology by using GenBank to lambda L chain and pSHC revealed sequence homology to IgG sequences from various species. The L chain cDNA contained the full translation sequence and 5' and 3' nontranslating region while the H chain cDNA coded for the secreted form of IgG1 and lacked sequences upstream from the C region. The derived amino acid sequences showed significant homology with various Ig sequences already described for human, mouse, rabbit, pig, and chicken but the degree of homology showed no consistency with established phylogenetic relationships.     

家兔BMP7基因的克隆及其生物信息学分析

在对已知部分编码序列(CDS)进行分析的基础上, 采用RT-PCR分步扩增以及RACE方法, 对家兔BMP7基因3′和5′末端未知序列进行了克隆与生物信息学分析。测序结果综合分析表明, 所获序列共计1 654 bp, 包括家兔BMP7近全长前肽、全长成熟肽CDS及3′非翻译序列(3′UTR), 将已有的序列向5′和3′端分别延伸了395 bp和628 bp。序列对比表明, 克隆的家兔BMP7 CDS部分与人、小鼠的对应序列的同源性分别为91.89%和89.32%, 预测的氨基酸序列同源性分别为96.51%和96.01%。家兔BMP7 3′UTR长446 bp, 与人、小鼠对应序列同源性分别为57.38%和45.57%; 具有2个转录终止信号位点。推测家兔BMP7成熟蛋白有BMPs特有的7个位置固定的半胱氨酸残基和TGF-β家族指纹。家兔BMP7 3′UTR区转录终止信号的可选择性可能与基因转录后调控有关。     

Sequences of mouse immunoglobulin light chain genes before and after somatic changes.

We have determined the nucleotide sequences of the germ line gene as well as a corresponding somatically mutated and rearranged gene coding for a mouse immunoglobulin lambdaI type light chain. These sequencing studies were carried out on three Eco RI-DNA fragments which had been cloned from BALB/c mouse embryos or a lambdaI chainsecreting myeloma, H2020. The embryonic DNA clone Ig 99lambda contains two protein-encoding segments, one for the majority of the hydrophobic leader (L) and the other for the rest of the leader and the variable (V) region of the lambda0 chain (Cohn et al., 1974); these segments are separated by a 93 base pair (bp) intervening sequence (I-small). The coding of the V region ends with His at residue 97. The second embryonic DNA clone Ig 25lambda includes a 39 bp DNA segment (J) coding for the rest of the conventionally defined V region (that is, up to residue 110), and also contains the sequence coding for the constant (C) region approximately 1250 untranslated bp (I-large) away from the J sequence. The J sequence is directly linked with the V-coding sequence in the myeloma DNA clone, Ig 303lambda, which has the various DNA segments arranged in the following order: 5' untranslated region, L, l-small, V linked with J, l-large, C, 3' untranslated sequence. The lg 303lambda V DNA sequence codes for the V region synthesized by the H2020 myeloma and is different from the lg 99lambda V DNA sequence by only two bases. No silent base change was observed between the two DNA clones for the entire sequence spanning the 5' untranslated regions and the V-coding segments. These results confirm the previously drawn conclusion that an active complete lambdaI gene arises by somatic recombination that takes place at the ends of the V-coding DNA segment and the J sequence. No sequence homology was observed at or near the sites of the recombination.     

Isolation of a genomal clone containing chicken histone genes.

We have used enriched chicken histone cDNA to select genomal clones from a chicken library. Because the cDNA probe also contained other sequences, a further screening of positive plagues with negative probes eliminated most non-histone gene clones. One 'positively-selected' genomal clone, lambda CH-01, hybridised with cloned sea-urchin histone genes and also detected histone genes in EcoRI-digested genomal sea-urchin DNA. Limited DNA sequencing of HaeIII fragments identified two sequences within the coding region of chicken histone H2A. A third fragment predicted an amino acid sequence with strong homology to an H1 histone sequence.     

Using iodinated single-stranded M13 probes to facilitate rapid DNA sequence analysis--nucleotide sequence of a mouse lysine tRNA gene.

From a recombinant lambda phage, we have determined a 387 bp sequence containing a mouse lysine tRNA gene. The putative lys tRNA (anticodon UUU) differs from rabbit liver lys tRNA at five positions. The flanking regions of the mouse gene are not generally homologous to published human and Drosophila lys tRNA genes. However, the mouse gene contains a 14 bp region comprising 13 A-T base pairs, 30-44 bp from the 5' end of the coding region. Cognate A-T rich regions are present in human and Drosophila genes. The coding region is flanked by two 11 bp direct repeats, similar to those associated with alu family sequences. The sequence was determined by a "walking" protocol that employs, as a novel feature, iodinated single-stranded M13 probes to identify M13 subclones which contain sequences partially overlapping and contiguous to an initially determined sequence. The probes can also be used to screen lambda phage and in Southern and dot blot experiments.     

Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP-dependent protein kinase. A lambda gt-11 cDNA library constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1, was screened using a 1.5-kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, lambda CAT4 and lambda CAT3, apparently encode two forms of C subunit designated C alpha and C beta, respectively. The nucleotide sequence of the C alpha and C beta cDNAs revealed differences in the coding region and particularly in the 3' untranslated region. However, the deducted amino acid sequences of C alpha and C beta subunits were 96% homologous to the sequences so far determined. Specific probes from the 3' coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC-PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the C alpha probe while the C beta probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the C alpha or C beta probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC-PK1 cells.     

Molecular cloning and nucleotide sequence of human pancreatic prechymotrypsinogen cDNA

The cDNA clone encoding human prechymotrypsinogen was isolated from a human pancreas cDNA library and its nucleotide sequence was determined. The sequence consists of a 16 bp 5' non-coding region, a 789 bp amino acid coding region and a 60 bp 3' non-coding region. The predicted product consists of 263 amino acids, including 18 amino acids for a signal peptide and 15 amino acids possible for an activation peptide. Southern blot analyses using the cloned cDNA as a probe revealed that human genomic DNA carries at least two genes that are related to chymotrypsinogen.     

白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析

目的 克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列.进一步推测这两个基因编码的蛋白,并分析其特征.方法 从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA.利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段.然后对已知片段进行3' RACE和5'RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列.利用相关软件对获得序列进行翻译和分析.结果 首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列.该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9％,与小鼠的相似度为82.7％.TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8％,与小鼠的相似度为86.6％.该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2％,与小鼠的相似度为81.9％.TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2％,与小鼠的一致度为84.4％.结论 本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征.     

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.     

Derivation of the sequence of the signal peptide in human C4b-binding protein and interspecies cross-hybridisation of the C4bp cDNA sequence

A 5' cDNA clone coding for human C4b-binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1-32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133-141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.