Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase

A series of cDNA clones corresponding to 1494 bases of rat muscle creatine kinase mRNA has been isolated and characterized. The identity of these clones has been confirmed by DNA sequence analysis and by comparison of the predicted amino acid sequence with that determined for the purified protein. The cDNA sequence accounts for the entire coding sequence of the creatine kinase protein in addition to the complete 3' untranslated region and 68 bases of 5' noncoding region. Sequences corresponding to the active site region of the protein, the initiation codon, the termination codon, and poly(A) addition signal have been identified.     

Putative high mobility group (HMG) non-histone chromosomal proteins from wheat germ. Isolation, characterisation and partial sequence analysis

Four proteins have been isolated from wheat germ by methods analogous to those used to isolate HMG proteins from animal tissue. All four proteins have been shown to be chromosomal in origin. Although amino acid analyses show that three of these proteins have compositions similar to those of the mammalian HMG proteins, N-terminal sequence analyses of these proteins show an absence of sequence homology with any of the mammalian HMG proteins.     

Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction

Summary The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.     

Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease

We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene.     

Serological analysis of species specificity in the high mobility group chromosomal proteins.

The non-histone chromosomal protein of the high mobility group (HMG-1) present in mouse liver was purified to homogeneity. Antibodies against this protein as well as pure HMG-1 derived from calf thymus and HMG-E purified from duck erythrocytes were elicited in rabbits. The interaction between the antibodies and the immunogens was measured by passive hemoagglutination and by quantitative microcomplement fixation. Quantitative microcomplement fixation assays revealed that the immunological distance between HMG-1 from calf thymus and HMG-1 from mouse liver and duck erythrocytes was 15. This corresponds to 3% sequence differences. It was estimated that amino acid substitution occurred at about seven positions in the polypeptide chain. Thus, HMG-1 proteins display remarkable evolutionary conservation in their primary sequence, similar to that displayed by histones H4 and H3, suggesting that their biological function is dependent on stringent structural requirements. HMG-E protein is significantly different from both HMG-1 and HMG-2 derived from calf thymus. As such, it is a protein unique to avian erythrocytes.     

Comparison of the high mobility group proteins and their chromatin distribution in trout testis and liver

The trout testis contains two major high mobility group (HMG) proteins HMG-T and H6 which, although related to the four mammalian HMGs, exhibit distinct variation as evidenced by differences in electrophoretic mobility and amino acid sequence. Previous work using various endonucleases as probes has shown that HMG-T and H6 are located at specific sites in the testis chromatin. The differentiation of testis cells during spermatogenesis is characterized by a unique transition from a histone-packaged genome to one bound by a class of small molecular weight, highly basic proteins, the protamines. Questions arise as to whether any of the HMG variability may be unique to the process of spermatogenesis and whether the histone-protamine transition occurring in most testis cells affects the HMG protein distribution and/or the specificity of the probe. In an attempt to answer these questions, the distribution of the HMG proteins in the chromatin of trout liver, a tissue lacking protamine, has been studied and comparisons made with testis. Liver HMGs exhibit the same electrophoretic characteristics as the testis HMGs indicating that the variability when compared to mammalian HMGs is primarily phylogenetic in origin rather than tissue-specific. Furthermore, micrococcal nuclease digestion of liver nuclei and its effect on the subsequent HMG protein distribution during chromatin fractionation yields a pattern very similar to that for testis, suggesting that the interaction of the HMGs with the remaining testis nucleohistone is not significantly altered by the ongoing transition to nucleoprotamine. Finally, the HMGs represent an unusually high proportion of the total testis non-histone protein population; the implications of this are discussed.     

Mouse glandular kallikrein genes. Nucleotide sequence of cloned cDNA coding for a member of the kallikrein arginyl esteropeptidase group of serine proteases

A library of cloned cDNA to male mouse submaxillary gland poly(A)-containing RNA was constructed in the plasmid pBR322. Inserts containing sequences estimated to be in the 1-5% abundance class were identified by hybridization to radiolabeled cDNA and examined by nucleotide sequence analysis. A sequence coding for a peptide with 57% homology to the only complete kallikrein sequence reported to date (from pig pancreas) was identified by a computer search program. This insert appears to code for the COOH-terminal 149 amino acids of a protein presumed therefore to be a serine protease. Comparison of the predicted amino acid sequence of this protein with analogous sequences in the three characterized members of the mouse submaxillary gland kallikrein arginyl esteropeptidase group of enzymes revealed extensive homology, although not complete identity. Thus, there are at least four members of this enzyme family expressed in the mouse submaxillary gland.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Distribution of high mobility group proteins in different tissues of rats during aging

The distribution of high mobility group (HMG) proteins has been studied in the liver, brain, kidney, lung, spleen, testis, thymus, and heart of young (19 weeks) and old (118 weeks) rats. These proteins were extracted with perchloric acid, fractionated by CM-Sephadex column chromatography, and analysed by acetic acid-urea polyacrylamide slab gel electrophoresis. As compared with that in young rats, the level of total HMG proteins in the old increased in liver and lung, decreased in thymus, heart, brain, and kidney, and remained unchanged in spleen and testis. In particular, the levels of HMG 1 and 2 were maximum in the thymus of young rats and dropped drastically in the old. However, the amount of HMG 17 was high in the spleen of both young and old rats, though it was comparatively higher in the former. Such age-dependent variation in the level of HMG proteins of different tissues denotes indirectly differences in the functional state of chromatin, and in growth and activity of cells, during aging.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Stimulation of V(D)J cleavage by high mobility group proteins.

V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 bp between the conserved heptamer and nonamer elements. The RAG1 and RAG2 proteins initiate the reaction by making double-strand DNA breaks at both signals, and must thus be able to operate on these two different spatial arrangements. We show that the DNA-bending proteins HMG1 and HMG2 stimulate cleavage and RAG protein binding at the 23 bp spacer signal. These findings suggest that DNA bending is important for bridging the longer spacer, and explain how a similar array of RAG proteins could accommodate a signal with either a 12 or a 23 bp spacer. An additional effect of HMG proteins is to stimulate coupled cleavage greatly when both signal sequences are present, suggesting that these proteins also aid the formation of a synaptic complex.