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 sequences of rabbit kappa light chains of b4 and b5 allotypes differ more in their constant regions than in their 3'' untranslated regions.

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.     

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.     

Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison

Immunoglobulin class switch involves a unique recombination event that takes place at the switch (S) region which is located 5' to each constant region (C) gene of the heavy (H) chain. For example, differentiation of the B lymphocyte from a mu-chain producer to an epsilon-chain producer is mediated by the switch recombination between the S mu and S epsilon regions. In order to elucidate the molecular mechanism for the switch recombination, we have determined nucleotide sequences surrounding the class switch recombination sites of the C epsilon and C gamma 3 genes and those in the 5' flanking regions of the C gamma 2a and C delta genes. The results indicate that the 5' flanking regions of all the CH genes except for the C delta gene contain the S regions which comprise tandem repetition of short unit sequences in agreement with the previous analyses of the S gamma 1, S gamma 2b, S mu, and S alpha regions. Comparison of the nucleotide sequences of all the S regions revealed that length as well as nucleotide sequences of the S regions vary among different classes of the CH gene, but they share short common sequences, (G)AGCT and TGGG(G). The nucleotide sequence of the S mu region is homologous to those of the other S regions in the decreasing order of the S epsilon, S alpha, S gamma 3, and (S gamma 1, S gamma 2b, s gamma 2a) regions. We have compared the nucleotide sequences immediately adjacent to the recombination sites of seven rearranged genes and have always fund tetranucleotides TGAG and/or TGGG, except for one case. Such tetranucleotides may constitute a part of the recognition sequence of a putative recombinase. These results provide further support for our previous proposal that the switch recombination may be facilitated by short common sequences dispersed in all the S regions.     

A mouse immunoglobulin heavy chain deletion mutant: isolation of a cDNA clone and sequence analysis of the mRNA

The mouse cell line IF2 secretes an immunoglobulin heavy chain lacking the CH1 domain. We have isolated and characterised a recombinant plasmid containing cDNA copies of the IF2 mutant mRNA. The cloned sequence extends from the nucleotides coding for amino acid 96 in the variable region through 100 nucleotides of untranslated region at the 3' end. The sequence of the cDNA insert reveals no discontinuity at the variable-hinge region junction, the site of the CH1 deletion. Experiments employing direct priming on the poly(A) tail of the IF2 heavy chain mRNA suggest that the 3' end of the cDNA clone (sequence C-C-C-T-G-C) is also the 3' end of the mRNA.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

Nucleotide sequence analysis of the cloned salmon preproinsulin cDNA

A cDNA library was constructed using polyadenylated RNA from salmon (Oncorhynchus keta) Brockmann bodies, plasmid vector pBR322, and in vitro recombinant DNA techniques. Insulin-related clones were identified with a cDNA probe generated from the same RNA and enriched for insulin sequences. Two recombinants were shown to contain the nucleotide sequence of the entire coding region and parts of the 5' and 3' untranslated regions. The salmon preproinsulin mRNA is about 760 nucleotides long, 315 of which code for the protein, while about 190 and 200 nucleotides belong to the 5' and 3' flanking regions, respectively. Comparison of the nucleotide sequences of salmon insulin mRNA with those from other species reveals that sequence conservation is limited to the regions coding for the B and A peptides and two segments of the signal peptide. The C-peptide region exhibits no significant sequence homology with the C-peptides of other vertebrates. The 5' and 3' untranslated regions of the salmon preproinsulin mRNA are homologous only with the anglerfish mRNA, whereas there is no evident homology with those of birds and mammals. In addition to establishing the sequence of the preproinsulin mRNA, cloned salmon insulin cDNA provides a specific probe for the analysis and isolation of genomic DNA fragments containing insulin genes.     

Complete nucleotide sequence of mouse immunoglobulin mu gene and comparison with other immunoglobulin heavy chain genes

We have determined the complete nucleotides sequence (2168 bases) of the immunoglobulin mu gene cloned from newborn mouse DNA. The cloned 13kb fragment contained the entire constant region gene sequence that is interrupted by three intervening sequences at the junction of domains as previously shown in the gamma 1, gamma 2 b and alpha genes. The amino acid sequence predicted by the nucleotide sequence agrees with that of the mu chain secreted by a myeloma MOPC104E except for 8 residues out of 448 residues. The homologous domains of the mu, gamma 1 and gamma 2b genes are more similar to each other than the different domains of the mu genes are. The result implicates that the class of the immunoglobulin heavy chain genes diverged after the heavy chain genes established the multi-domain structure. The short intervening sequences of the mu and gamma genes are more conserved than the coding sequences except for the COOH-terminal domains. The results implicate that the nucleotide sequence of the intervening sequence is under selective pressure, possibly to maintain a secondary structure of the nuclear RNA to be spliced.     

The complete sequence of the human beta-myosin heavy chain gene and a comparative analysis of its product

We have isolated and sequenced the gene and the cDNA coding for the human cardiac beta-myosin heavy chain (designated MYH7). The gene is 22,883 bp long. The 1935 amino acids of this protein (Mr223,111) are encoded by 38 exons. The 5' untranslated region (86 bp) is split by two introns. The 3' untranslated region is 114 bp long. Three Alu repeats were identified within the gene and a fourth one in the 3' flanking intergenic region. The molecular organization of this gene reflects the conservative pattern with respect to size, coding ratio, and number or position of introns characteristic of vertebrate sarcomeric myosin heavy chain genes. The protein sequence of the human beta-heavy chain was compared with corresponding (homologous) sequences of rabbit, rat, and hamster as well as with the (heterologous) embryonic heavy chain sequences of rat, chicken, and man. The results show that protein subregions responsible for basic functions of myosin heavy chains (nucleotide binding and actin binding) are very similar in homologous and heterologous heavy chains. Regions that differ in their primary sequences in heterologous heavy chains appear to be highly conserved within mammalian beta-myosin heavy chains. Constant and variable subregions of heavy chains are discussed in terms of functional significance and evolutionary relatedness.     

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

cDNA clones encoding bovine gamma-crystallins

We have determined the nucleotide sequence of two bovine lens gamma-crystallin cDNA clones, pBL gamma II-1 and pBL gamma III-1. The 644 bp cDNA insert of pBL gamma II-1 contains coding information for the entire amino acid sequence of bovine gamma II-crystallin. The 497 bp cDNA insert of pBL gamma III-1 encodes a homologous but different gamma-crystallin polypeptide, and appears to lack the coding information for the C-terminal 17 amino acid residues. While the nucleotide and predicted amino acid sequences of the coding regions of the clones show a high degree of homology, the untranslated leader sequences are relatively dissimilar. The leader sequence of pBL gamma III-1 is strikingly homologous to a portion of a rabbit immunoglobulin alpha-heavy chain mRNA.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Mouse heavy chain variable regions: nucleotide sequence of a germ-line VH gene segment

We have constructed a library of Balb/c mouse embryo DNA in the vector Charon 4A. The library was searched for sequences homologous to the VH region of a cloned cDNA of the UPC10 heavy chain mRNA. In this paper, we describe the structure and the partial nucleotide sequence of one of such clones (VH441). The nucleotide sequence of this germ-line gene indicates that it encodes amino-acids 1-98 of the X44 and J601 galactan-binding VH regions, but that it differs from the UPC10 VH segment by four single base changes. The VH gene appears to contain a 101 bases long intervening sequence within a precursor sequence identical to the precursor sequence of UPC10. The 3' non coding sequence of the V gene contains the two conserved sequences found in embryonic V DNA segments, CACAGTG and ACATGAACC, separated by 23 nucleotides and a sequence CACTGTG separated by 33 nucleotides from the first heptamer.     

Characterization and nucleotide sequence of the gene for the common alpha subunit of the bovine pituitary glycoprotein hormones.

The gene coding for the common alpha subunit of the bovine pituitary glycoprotein hormones was isolated from a bovine genomic library. The gene spans roughly 16.5 kbp, contains three intervening sequences, and codes for a message of approximately 730 nucleotides. The complete coding region of the gene was sequenced as well as 315 nucleotides of 5' flanking sequence and the entire intron C. Only a single base difference was found when the sequence of the gene was compared with that of the cDNA. Genomic blotting experiments suggest the presence of a single alpha subunit gene. Comparison of the bovine and human alpha subunit genes indicated that the high level of homology observed in the coding regions has been maintained throughout the 5' and 3' untranslated regions, and at least 90 nucleotides of the 5'flanking regions. Additionally, there is an 18 base pair sequence present in both the 5' flanking and 5' untranslated regions of the gene that is homologous to a region of the chick ovalbumin gene. This ovalbumin sequence has been suggested as a binding site for the progesterone receptor-complex.     

Analyses of the splenic mRNA expressed by rabbits of different immunoglobulin kappa-light chain allotypes: conserved sequences in the 3' untranslated region and allotype-specific probes

The allelism of the structural genes for the complex rabbit b allotypes of immunoglobulin kappa-light chains has been questioned because of observations of unexpected phenotypic expression of "latent" allotypes. We find that the coding sequences of the b4 and b5 "alleles" are only 80% homologous for the last 60 nucleotides but there is a high degree of homology (96%) in the 3' untranslated region (3'DT). The high conservation of 3' DT region sequences enabled us to detect kappa-light chain mRNAs from rabbits of different genetic types (b4, b5, b9 and bbas) on northern blots and dot blots. We can distinguish mRNA encoding b9 and b5 allotypes on dot blots with b5 fragment-probes of known sequence and detect mRNA produced by unstimulated cultured splenic lymphocytes. Analyses of mRNA from cultured cells manipulated to enhance mRNA synthesis and production of unexpected or "latent" b allotypes can now be conducted.     

Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.