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At least 721 records · Page 40Linked to original sources
PubMed1984

Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones.

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.

Animals↗
PubMed1985

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme.

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.

Amino Acid Sequence↗
PubMed1987

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin.

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.

Amino Acid Sequence↗
PubMed1988

Melting of a self-complementary DNA minicircle. Comparison of optical melting theory with exchange broadening of the nuclear magnetic resonance spectrum.

Melting curves are calculated for the 16-base-pair duplex DNA sequence 5' GTATCCGTACGGATAC 3' linked on the ends by TTTT single-strand loops. The equilibrium statistical thermodynamic theory of DNA melting is modified to include effects of end-loops on the melting transition. An excellent fit of the experimental melting curve in 0.2 M-NaCl is obtained using two adjustable parameters, one for end-loop formation and the other for formation of the complete 40-base single-strand loop. The best-fit calculated melting curve permits evaluation of these parameters. The free energy to close a TTTT end-loop is 2.12 kcal/mol (1 cal = 4.184 J). A TTTT end-loop or hairpin loop is significantly more stable than an internal loop of comparable size sandwiched between two helical regions, even after allowing for the different stacking contributions. Reasons for this increased stability are presented. The loop free energy of the 40-base single-strand open minicircle is evaluated to be +1.27 kcal/mol, thus favoring the melting of two end-loops into the large open minicircle. The present results are compared with those of others for d(T-A) oligomers. The sequence TTTT forms a more stable end-loop, or hairpin, than TATA by about 2.0 kcal/mol. Theoretical rate constants for the proton-transfer step in the standard hydrogen-exchange model are calculated by extending the theory of diffusion-controlled reactions to take account of the electrostatic potential of the DNA. The predicted ratios of rate constants for different pairs of catalysts exchanging an A.T proton agree satisfactorily with the available experimental data for a 14-base-pair linear duplex, which confirms the diffusion-control of the proton-transfer step. Data presented here for the 16 base-pair duplex of the minicircle are consistent with catalysis-limited exchange in which the proton-transfer step is likewise diffusion-controlled. Under catalysis-limited conditions, the imino proton exchange rates are predicted from the catalytic rate constants, prevailing buffer catalyst concentrations, and the equilibrium constants to form the unstacked open state of optical melting theory. The observed exchange rates of the A.T base-pairs show no sign of the strong predicted end-melting trend, and exceed the predicted values by factors of 10 to 400. Moreover, the succession of "melting" in the nuclear magnetic resonance line-broadening deviates from that predicted by optical melting theory.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1992

Complementary DNA synthesis in situ: methods and applications.

In situ transcription is the synthesis of cDNA within cells. This chapter has illustrated some of the application of IST to the study of gene expression in complex cell environments. While the importance of transcription in modulating cellular activity has been long appreciated, the role of translational control mechanisms in regulating central nervous system functioning is just beginning to be recognized. Previous limitations in the availability of tissue have made it difficult to construct cDNA libraries from defined cell populations, to examine translational control, and to quantitate differences in the amount of mRNA for many distinct mRNAs in the same sample. In situ transcription facilitates all of these procedures, making it possible to characterize aspects of gene regulation that were previously difficult. Indeed, taken to its furthest extreme it is now possible to characterize gene expression in single live cells. This level of analysis allows basic questions, such as How different morphologically identical cells are at the level of gene expression, and How synaptic connectivity and glial interactions influence gene expression in single cells, to be experimentally approached. The ability to characterize gene expression in small amounts of tissue and single cells is critical to gaining an understanding of the contribution of specific cell types to the physiology of the central nervous system.

Animals↗