Cloning, characterization, and expression in Escherichia coli of three creatine kinase muscle isoenzyme cDNAs from carp (Cyprinus carpio) striated muscle

In vertebrates, the creatine kinase isoenzyme family consists of four types of isoforms: cytosolic muscle type (M-CK), cytosolic brain type (B-CK), mitochondrial ubiquitous, acidic type (Miu-CK), and mitochondrial sarcomeric, basic type (Mis-CK). Until recently, the existence of more than one subisoform of CK isoenzyme has been demonstrated only in fishes by starch gel electrophoresis. We report herein the isolation of three full-length cDNAs that correspond to three closely related creatine kinase M-CK genes from common carp (Cyprinus carpio), designated the M1-CK, M2-CK, and M3-CK genes. Using oligonucleotide probes that correspond to the same region but with the most variable sequences, different restricted genomic hybridization patterns have been obtained. These Southern blot results indicate that the three cDNAs come from different genes. Northern blot analysis using probes that correspond to the 3'-untranslated regions further show that all three subisoforms are expressed specifically in carp muscle. The deduced amino acid sequences of these three subisoforms of carp M-CK show about 85% identity to mammalian M-CK isoenzyme. Finally, the three cDNAs have been expressed in Escherichia coli with a molecular mass of approximately 43,000 Da, and these recombinant proteins exhibit creatine kinase activity. All of these data suggest that the M-CK isoenzymes have at least three subisoforms in carp.     

Cloning and sequence analysis of two cDNAs encoding cyclin A and cyclin B in the zebra mussel Dreissena polymorpha

Cyclins are key components in the progression of both mitotic and meiotic cell cycle control. Full-length cDNA clones encoding cyclin A and cyclin B were isolated from a zebra mussel testis cDNA library. The clones contained open reading frames of 419 and 434 amino acids, had similarity to cyclins A and B from other species, but also some unique features in their sequences. Cyclin A and B mRNA was expressed in testis, ovary, gill, mantle, muscle, and eggs, as shown by specific polymerase chain reaction.     

Insulin synthesis in chick embryo retinas during development

Retinas of chick embryos contain insulin and further, are capable of synthesizing it, as demonstrated by incubating retinas at different ages (7th-18th day) with [3H]leucine. The synthesized radioactive insulin was isolated and assayed by means of a HPLC procedure. The synthesis of insulin was found to be highest in the youngest retinas studied (day 7), afterwards it declined with age except for an increment found at 14-15 day. Explants of chick embryo retinas, cultured in vitro, rapidly degraded insulin. Nevertheless, the content of immunoreactive insulin in retinal explants diminished slowly with the age of culture, so that, after 8 days of incubation, it was about 60% of the content found in the retinas at the beginning of incubation. This was proof that cultured explants are capable of efficiently synthesizing insulin. The synthesized [3H]insulin was released from explants into the medium. This was evident also after 6-8 days in culture.     

Heart rate during development in the turtle embryo: effect of temperature

Growth and development can occur over a wide range of physical conditions in reptiles. Cardiovascular function must be critical to this ability. However, information on cardiovascular function in developing reptiles is lacking. Previous work indicated that in reptiles the effects of temperature on growth and metabolism are largely restricted to early development. This study examined whether the previously observed effects of temperature and different perinatal patterns of metabolism observed in amniotic vertebrates are correlated with cardiovascular function. Embryonic and hatchling carcass mass, heart mass and heart rate (HR) were compared for snapping turtle eggs (Chelydra serpentina) incubated at 24 degrees and 29 degrees C. Incubation time was shorter at 29 degrees C (56.2 days) than at 24 degrees C (71.1 days). Carcass and heart growth showed a sigmoidal pattern at both temperatures. However, cardiac growth showed a relative decrease as incubation proceeded. Incubation temperature significantly affected the HR pattern during development. The HR of embryos incubated at 24 degrees C was constant for most of incubation (51.8 +/- 4.8 min-1). A small decrease was observed just prior to and a large decrease immediately following hatching (posthatch, 22.3 +/- 4.1 min-1). At 29 degrees C embryonic HR was greater than at 24 degrees C early in development (72.3 +/- 3 min-1). The HR steadily decreased to values equivalent to those at 24 degrees C. The HRs of 24 degrees C and 29 degrees C hatchlings were not different. Cardiac output (estimated as the product of heart mass and HR) increased rapidly during early development and then slowed dramatically at both temperatures. These data are consistent with the suggestion that temperature exerts its effects primarily early in development. Furthermore, the changes in cardiovascular function are correlated with metabolic changes in hatching vertebrates.     

Characterization of two cDNAs encoding auxin-binding proteins in Nicotiana tabacum

The isolation and the characterization of two tobacco cDNAs, Nt-ERabp1 and Nt-ERabp2, homologous to Zm-ERabp1, encoding the major auxin-binding protein from maize coleoptiles, are described. Their predicted amino acid sequences correspond to proteins of ca. 21 kDa, in which the characteristic regions common to ABP1-related polypeptides are well-conserved. Southern analysis indicates that the genes corresponding to Nt-ERabp1 cDNA and Nt-ERabp2 cDNA derive respectively from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid progenitors of Nicotiana tabacum. Analysis of mRNA distribution in tobacco plants indicates that these two genes are preferentially expressed in flowers and growing seedlings. Whatever the tissue tested, Nt-ERabp1 mRNA is more abundant than Nt-ERabp2 mRNA. Furthermore, RT-PCR reveals developmental and organ-specific expression of these two genes in flower parts of tobacco plants. In particular, regulation of Nt-ERabp1 mRNA accumulation appears to be correlated with elongation growth of each floral organ. Recombinant Nt-ERabp1, produced in Escherichia coli, is recognized by antibodies raised against Zm-ERabp1.     

Cloning of two cDNAs encoding calnexin-like and calreticulin-like proteins from maize (Zea mays) leaves: identification of potential calcium-binding domains

The U-turn (uridine turn) is an RNA structural motif that contains a change in backbone direction stabilized by specific interactions across the bend. It was first identified in the anticodon loop and the T-loop of yeast tRNA(Phe) (Quigley & Rich, 1976, Science 194:796-806) and has recently also been found in the crystal structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB, 1994a, Nature 372:68-74). These U-turn motifs follow a UNR consensus sequence (where N is any nucleotide and R is G or A). Here we report that the frequently occurring GNRA tetraloops also contain a U-turn motif, and we discuss the role of U-turns as abundant tertiary structural motifs in RNA.     

Cloning and sequence analysis of cDNAs encoding precursors of sarafotoxins. Evidence for an unusual "rosary-type" organization

Sarafotoxins (SRTXs) are 21-amino acid peptides structurally and functionally similar to endothelins (ETs). To understand how SRTXs are overproduced in venom glands of the snakes Atractaspis engaddensis and hence used as toxins, we cloned cDNAs encoding SRTXs and elucidated their nucleotide sequences. We predict that SRTX precursors are large prepropolypeptide chains with an unusual "rosary-type" structure made of 12 successive similar stretches of 40 residues (39 in the first stretch). Each stretch begins with a "spacer" of 19 invariant residues (18 in the first stretch) immediately followed by the sequence of one SRTX isoform. Six different isoforms are identified within a single precursor molecule. Maturation of the precursor may require endopeptidases that cleave the Leu-Cys bond and the Trp-Arg/Lys bond invariably found at the SRTX N and C termini, respectively.     

Cloning and overexpression of two cDNAs encoding the low-CO2-inducible chloroplast envelope protein LIP-36 from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a unicellular green alga, grows photoautotrophically at very low concentrations of inorganic carbon due to the presence of an inducible CO2-concentrating mechanism. During the induction of the CO2-concentrating mechanism at low-CO2 growth conditions, at least five polypeptides that are either absent or present in low amounts in cells grown on high-CO2 concentrations are induced. One of these induced polypeptides with a molecular mass of 36 kD, LIP-36, has been localized to the chloroplast envelope. The protein was purified and the partial internal amino acid sequences were obtained through lys-C digestion. Two cDNAs encoding LIP-36 have been cloned using degenerate primers based on the amino acid sequences. The two genes encoding LIP-36 are highly homologous in the coding region but are completely different in the 5'-end and 3'-end untranslated regions. The deduced protein sequences show strong homology to the mitochondrial carrier protein superfamily, suggesting that LIP-36 is a chloroplast carrier protein. The regulation of the expression of these two genes at high- and low-CO2 growth conditions is also different. Both genes were highly expressed under low-CO2 growth conditions, with the steady-state level of LIP-36 G1 mRNA more abundant. However, neither gene was expressed at high-CO2 growth conditions. The gene products of both clones expressed in Escherichia coli were recognized by an antibody raised against LIP-36, confirming that the two cDNAs indeed encode the C. reinhardtii chloroplast envelope carrier protein LIP-36.