Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos

A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.     

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.     

Human hTM alpha gene: expression in muscle and nonmuscle tissue.

We have isolated a cDNA clone from a human skeletal muscle library which contains the complete protein-coding sequence of a skeletal muscle alpha-tropomyosin. This cDNA sequence defines a fourth human tropomyosin gene, the hTM alpha gene, which is distinct from the hTMnm gene encoding a closely related isoform of skeletal muscle alpha-tropomyosin. In cultured human fibroblasts, the hTM alpha gene encodes both skeletal-muscle- and smooth-muscle-type alpha-tropomyosins by using an alternative mRNA-splicing mechanism.     

Differential expression of two skeletal muscle beta-tropomyosin mRNAs during Xenopus laevis development

A cDNA clone for a Xenopus laevis skeletal muscle beta-tropomyosin (beta-TMad) isoform was isolated from an adult skeletal muscle cDNA library. Sequence analysis revealed that this clone corresponded to a second beta-tropomyosin mRNA distinct from the one that was previously characterized (beta-TMemb). The two skeletal beta-TM mRNAs originate from distinct genes and are differentially expressed during development. Beta-TMemb mRNA is expressed only in the somites of the early embryo while beta-TMad mRNA is expressed in pre-metamorphic tadpoles and adult skeletal muscles. We have isolated the promoter region of the beta-TMemb gene and shown that a DNA construct containing 2.9 kb of promoter region is properly expressed after injection in the embryo.     

The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor.

Two different cDNAs for human granulocyte colony-stimulating factor (G-CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G-CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G-CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G-CSF cDNA and the DNA fragment containing the G-CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G-CSF gene is interrupted by four introns and a comparison of the structures of the two G-CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5' splice donor sequences in the second intron of the G-CSF gene. When the G-CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.     

Three types of Gi alpha protein of the guinea-pig lung: cDNA cloning and analysis of their tissue distribution.

cDNA clones encoding three types of Gi alpha, the alpha subunit of GTP-binding protein (Gi1 alpha, Gi2 alpha, and Gi3 alpha), were isolated from a cDNA library of the guinea-pig lung. Nucleotide sequence analysis revealed a high degree of homology with other mammalian Gi alpha cDNAs. By RNA blot analysis, the expression pattern of Gi1 alpha was more tissue-specific than those of other types of Gi alphas in the guinea-pig tissues examined. While Gi2 alpha and Gi3 alpha mRNAs were ubiquitously expressed in all tissues examined, Gi1 alpha mRNA was mainly expressed in the brain, lung and kidney. These results suggest that each Gi alpha protein may have a different role.     

Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine-gated strychnine-sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent-human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2-p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.     

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

Molecular characterization of an anther-preferential gene from rice

Expression levels of anther-expressed genes in rice were estimated by plaque hybridization. A total of 33 cDNAs, isolated randomly from an anther-enriched cDNA library, were used as probes to hybridize both anther and leaf cDNAs. The expression level of individual cDNA clones was then estimated by counting the number of plaques hybridized to each probe. Based on abundance patterns that appeared in both anther and leaf cDNA libraries, the clones were classified into three groups. This classification showed that the majority of the clones (one group) exhibited expression in both cDNA libraries at almost equal frequency. The other two groups showed either low or no expression in the leaf cDNA library. Among the cDNA clones,RA1003 (detected only in the anther cDNA library) was selected and further characterized at the molecular level. Consistent with the results of the plaque hybridization experiment, northern blot analysis also revealed no gene expression in vegetative organs, leaves, or roots. However, expression was high in the flowers, especially in the anthers. Detailed molecular studies of the gene are also described and discussed here.