Nucleotide sequence of a cDNA clone encoding a Drosophila muscle tropomyosin II isoform

A cDNA clone was sequenced that contains the entire coding region for the muscle tropomyosin II isoform from Drosophila. The cDNA clone is 1253 nucleotides (nt) long and contains an 88-nt 5'-leader sequence and a 310-nt 3'-untranslated sequence. The muscle tropomyosin II isoform consists of 285 amino acids and is 60% homologous with the previously reported muscle tropomyosin I isoform Drosophila and 55% homologous with rabbit muscle tropomyosin.     

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Expression of a muscle-type alpha-actinin cDNA clone in non-muscle cells

We have previously isolated a chick smooth muscle-type alpha-actinin cDNA clone (C17) from a chick embryo fibroblast cDNA library. As part of an investigation into a possible role for a muscle isoform of alpha-actinin in non-muscle cells, we have cloned C17 into a eucaryotic expression vector, pKCR3, and examined the distribution of the expressed protein in non-muscle, monkey COS cells. We report here that the muscle isoform of chick alpha-actinin encoded by C17, was found in focal contacts and periodically distributed along actin filaments.     

A human placental cDNA clone that encodes nonhistone chromosomal protein HMG-1.

From a human placental lambda gt11 cDNA library, we have isolated a cDNA clone that encodes the entire 215-residue amino acid sequence of HMG-1. Analysis of an internal sequence similarity suggests that the DNA-binding domains of HMG-1 are separated by a rather long and flexible linker segment. Southern blotting of DNA digested with BamHI indicated a highly variable number of genes (or pseudogenes) for HMG-1 in different species. Characterization of HMG-1 mRNA expression by Northern blotting showed that three mRNA species of approximately 1.0, 1.4 and 2.4 kb were expressed in all mammalian organs and cell lines examined. These included several rat organs at different stages of development. Northern analysis also suggested the occurrence of HMG-1 mRNA in an invertebrate and a plant species.     

Isolation and characterization of cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform

cDNA clones encoding rat fibroblast tropomyosin 4 (TM-4) were isolated and characterized. DNA sequence analysis was carried out to determine the sequence of the protein. The derived amino acid sequence revealed that rat fibroblast TM-4 was found to contain 248 amino acids. The amino acid sequence of rat fibroblast TM-4 was compared with two other low molecular weight TM isoforms, equine platelet beta-TM and a human fibroblast TM. Rat TM-4 exhibited 98% sequence identity with the equine platelet TM but only 75% identity with the human fibroblast TM isoform. The high degree of conservation between the rat and equine proteins indicates that they belong to the same isotype of TM. Comparison of the amino acid sequences of the three low molecular TM isoforms along the length of the proteins reveals regions that are strongly conserved and regions that have considerably diverged. In the regions from amino acid residues 1 to 148 and 176 to 221, amino acid substitutes are moderate. The most variant regions in the sequence are in the middle part of the proteins from amino acids 149 to 175 and at the carboxyl-terminal region of the proteins from amino acids 222 to 248. The differences in the sequence of the rat and platelet TMs compared to the human TM may define distinct functional domains among the low molecular weight TMs. In addition, expression of tropomyosin was studied in a variety of tissues and transformed cells. We also demonstrate that at least three separate genes encode tropomyosins expressed in rat fibroblasts.     

Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase

A cDNA clone encoding the glyoxysomal enzyme isocitrate lyase (ICL) (EC 4.1.3.1) was isolated from a library prepared from cotton (Gossypium hirsutum L.) cotyledon poly(A)+ RNA. The clone is 1893 basepairs (bp) in length and contains a 1728 bp open reading frame encoding a polypeptide of 576 residues (Mr = 64,741). The deduced amino acid sequence of cotton ICL is 85.2%, 90.3% and 41.1% identical to ICL from rapeseed, castor bean and E. coli, respectively. Cotton ICL has a C-terminal tripeptide of A-R-M which is a putative trafficking signal for peroxisome (glyoxysome) proteins.     

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase

Summary Nitrate reductase (NR) assays revealed a bi-specific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)⁺ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%–77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch. These sequence data appeared in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X54097     

Characterization and sequence of a cDNA clone of gamma-glutamyltranspeptidase.

We have isolated several cDNA's complementary to gamma-glutamyltranspeptidase (GGT) mRNA by screening a rat kidney library constructed in lambda gtll with antibodies specifically reactive to the enzyme protein. The clone selected an mRNA that was translated into a 62 Kd peptide, corresponding to the GGT precursor. The longest clone isolated was 1842 bp long with an open reading frame coding for 565 amino acids. The length of the mRNA coding for GGT was estimated to be 2.2 kb long. The amino acid sequence derived from the nucleotide sequence matched the short sequences determined by us as well as by other authors.     

Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.

We isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an alpha fast-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle alpha-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle alpha-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle alpha-tropomyosin and the smooth muscle alpha-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.     

Cloning and expression analysis of a cDNA that encodes a leech hemerythrin

We report the cDNA sequence of a leech hemerythrin. A cDNA was isolated from a Theromyzon tessulatum cDNA library and encodes a 120 amino acid protein of about 14 kDa. The predicted protein contains the hemerythrin signature sequence and the iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin. The protein displayed the highest identity to myohemerythrin, a non-heme iron-binding protein described in sipunculids. Expression analysis indicated that the mRNA is widely expressed in leech and is stage specific in appearance, being absent after the two first blood meals, appearing after the last blood meal during the period preceding oogenesis and disappearing after egg laying.     

Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease.

The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinic-apyrimidinic (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Drosophila extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-kb mRNA also hybridized to the AP3 cDNA, but this species was restricted to the early stages of development.     

An insertion within a variably spliced Drosophila tropomyosin gene blocks accumulation of only one encoded isoform

We have characterized an aberrant allele of a variably spliced Drosophila tropomyosin gene. The allele was recovered from the flightless Ifm(3)3 strain, which has been shown to have structurally and functionally abnormal indirect flight muscles. The transcribed portion of the mutant gene is interrupted by an 8,8 kb insertion of middle repetitive DNA having a structure typical of copia-like Drosophila mobile elements. The insertion is positioned so as to interrupt an exon sequence in one splicing mode and, simultaneously, an intron in the alternate mode. As a consequence of the insertion the allele fails to direct synthesis of the flight muscle-specific tropomyosin isoform, but remains capable of specifying a second isoform, which accumulates in nonfibrillar Drosophila muscles.