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C B Green

Publications and source records attributed to C B Green.

32 records · Page 2Linked to original sources
PubMed1996

Use of a high stringency differential display screen for identification of retinal mRNAs that are regulated by a circadian clock.

We report here the initiation of a systematic screen to identify clock-controlled mRNAs from the retina of Xenopus laevis using mRNA differential display. Xenopus retina contains an endogenous circadian clock located within the photoreceptor layer. The retinal block controls many aspects of physiology, including gene transcription. This screen uses differential display, a PCR based procedure, to compare retinal mRNA populations at different times of day in constant darkness, for identification of messages that exhibit rhythmic expression. Out of approx. 2000 mRNAs that we have screened to date, we have identified four candidates for clock-controlled mRNAs. Initial characterization of one of these PCR products shows that it recognizes a pair of mRNA bands on Northern blots that exhibit high amplitude rhythms. This pair of messages is called RM1 and shows peak levels of expression in the subjective night. In situ hybridization shows that this clock-controlled message is specifically localized to the clock containing photoreceptor cell layer within the retina. Identification of new messages that are under the control of the circadian clock has broad relevance in retinal physiology and provides an opportunity to gain insight into the molecular mechanism of vertebrate circadian control.

Animals↗
PubMed1995

Regulation of tryptophan hydroxylase expression by a retinal circadian oscillator in vitro.

Many aspects of retinal physiology are controlled by a circadian clock including at least two steps in the melatonin synthetic pathway: the activity of the enzyme, N-acetyltransferase (NAT), and mRNA levels of the rate-limiting enzyme trytophan hydroxylase (TPH). Light and dopamine (through D2-like dopamine receptors) can phase shift the clock, and can also acutely inhibit NAT activity, resulting in supressed melatonin synthesis. In this paper, we show that eyecups cultured in constant darkness maintain a clock-controlled rhythm in TPH mRNA, with low levels in early day, rising to a peak in early night. Both eyecups and isolated retinas, cultured in light during the day, also exhibit a similar increase in TPH mRNA levels, indicating that this expression is not acutely inhibited by light. Treatment with light or quinpirole (D2 dopamine receptor agonist) in early night, at a time and dose that acutely inhibits NAT activity, does not change levels of TPH mRNA. Addition of eticlopride (D2 dopamine receptor antagonist) during the day, also has no effect on the normal daytime increase in TPH message levels. Therefore, TPH mRNA level is controlled by a circadian clock located within the eye, but acute effects of light or dopamine are not detected.

Animals↗
PubMed1995

Inhibition of translation initiation on Escherichia coli gnd mRNA by formation of a long-range secondary structure involving the ribosome binding site and the internal complementary sequence.

Previous research has indicated that the growth rate-dependent regulation of Escherichia coli gnd expression involves the internal complementary sequence (ICS), a negative control site that lies within the 6-phosphogluconate dehydrogenase coding sequence. To determine whether the ICS acts as a transcriptional operator or attenuator, we measured beta-galactosidase-specific activities in strains carrying gnd-lac operon and protein fusions containing or lacking the ICS. Whereas the presence of the ICS repressed beta-galactosidase expression from a protein fusion by 5-fold during growth on acetate and by 2.5-fold during growth on glucose, it had no effect on beta-galactosidase expression from an operon fusion. In vitro ribosome binding experiments employing the primer extension inhibition (toeprint) assay demonstrated that the presence of the ICS in gnd mRNA reduces both the maximum extent and the rate of ternary complex formation. Moreover, the effects of deletions scanning the ICS on in vivo gene expression were highly correlated with the effects of the deletions on ribosome binding in vitro. In addition, the distal end of the ICS element was found to contribute more to ICS function than did the proximal portion, which contains the complement to the Shine-Dalgarno sequence. Finally, RNA structure mapping experiments indicated that the presence of the ICS in gnd mRNA reduces the access of the nucleotides of the ribosome binding site to the single-strand-specific chemical reagents dimethyl sulfate and kethoxal. Taken together, these data support the hypothesis that the role of the ICS in the growth rate-dependent regulation of gnd expression is to sequester the translation initiation region into a long-range mRNA secondary structure that blocks ribosome binding and thereby reduces the frequency of translation initiation.

Acetates↗
PubMed1994

Tryptophan hydroxylase expression is regulated by a circadian clock in Xenopus laevis retina.

A circadian clock has been localized to the photoreceptor layer in the Xenopus laevis retina. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin synthesis in Xenopus laevis retina is tryptophan hydroxylase. A cDNA clone coding for Xenopus tryptophan hydroxylase was isolated, characterized, and used as a probe for analysis of tryptophan hydroxylase mRNA expression. Northern blot analyses of total retinal RNA show that the tryptophan hydroxylase message levels are low in the day and higher at night. The expression of tryptophan hydroxylase mRNA is under circadian control because rhythmic changes are also seen in constant darkness, with elevated levels during the subjective night. Nuclear run-on analysis during the first subjective day in constant darkness revealed that transcription initiation is low early in the day and increases throughout the day. Our observations suggest that the circadian clock modulates tryptophan hydroxylase gene expression. An understanding of how the circadian clock controls tryptophan hydroxylase expression may lead to a clearer understanding of the melatonin biosynthetic pathway, and possibly the clock itself.

Amino Acid Sequence↗
PubMed1993

Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active.

Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.

Amino Acid Sequence↗
PubMed1992

Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM).

The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic domains necessary for the two-step formation of alpha-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA were identified by examining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contained exon A, the 315-nucleotide (nt) segment situated between the PHM and PAL domains and present in rPAM-1 but absent from rPAM-2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment encoding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane protein; rPAM-3a lacks a 204-nt segment including the transmembrane domain and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the PHM domain; alternative splicing generates a novel 3'-region encoding a COOH-terminal pentapeptide followed by 1.1 kb of 3'-untranslated region. The soluble rPAM-5 protein lacks PAL, transmembrane, and cytoplasmic domains. These three forms of PAM mRNA can be generated by alternative splicing. The major forms of PAM mRNA in both lobes of the pituitary are rPAM-3b and rPAM-2. Despite the fact that anterior and neurointermediate pituitary contain a similar distribution of forms of PAM mRNA, the distribution of PAM proteins in the two lobes of the pituitary is quite different. Although integral membrane proteins similar to rPAM-2 and rPAM-3b are major components of anterior pituitary granules, the PAM proteins in the neurointermediate lobe have undergone more extensive endoproteolytic processing, and a 75-kDa protein containing both PHM and PAL domains predominates. The bifunctional PAM precursor undergoes tissue-specific endoproteolytic cleavage reminiscent of the processing of prohormones.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of PSP-I and PSP-II, two major proteins from porcine seminal plasma.

Two major glycoproteins, designated PSP-I and PSP-II, were purified from porcine seminal plasma by ammonium sulfate fractionation, CM-cellulose chromatography, gel filtration on Sephadex G-75 (superfine), and reverse phase high performance liquid chromatography. These two proteins exist in several forms differing mainly in the carbohydrate moiety. The complete amino acid sequence of PSP-I has been determined by automated Edman degradation of peptides generated by proteolytic digestion and cyanogen bromide cleavage. The protein is 109 residues long and has a single glycosylation site at the asparagine residue at position 47. In addition, the N-terminal sequence of PSP-II has also been determined. PSP-I is a unique protein; a sequence homology search using the protein data base did not reveal any significant homology with other proteins. PSP-II shares 50% sequence homology with a family of zona pellucida-binding glycoproteins at the N-terminus.

Amino Acid Sequence↗
PubMed1992

Expression of prohormone processing enzymes in neuroendocrine and non-neuroendocrine cells.

The biosynthesis of many peptides thought to have autocrine or paracrine effects on cell growth requires a series of enzymatic steps. The level of expression of two of these posttranslational processing enzymes was compared in several endocrine and non-neuroendocrine cell lines. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional copper- and ascorbate-dependent enzyme essential in the formation of alpha-amidated peptides. Carboxypeptidase H (CPH; EC 3.4.17.10) removes basic residues from the carboxyterminus of the products of endoproteolytic cleavage of prohormones and is generally essential in the formation of substrates for PAM. PAM messenger RNA (mRNA) and activity were detectable in both endocrine (AtT-20, GH3) and non-neuroendocrine (L, 3T3, COS, BRL, C127) cells. Except for BRL cells, CPH mRNA and enzymatic activity were detectable in all the cell lines. BRL cells contained no detectable CPH mRNA and had a different carboxypeptidase B-like activity. Thus, expression of secretory granule-associated processing enzymes is not limited to cells of a classic neuroendocrine phenotype.

Amino Acid Sequence↗
PubMed1990

Cloning and nucleotide sequence analysis of the human beta-microseminoprotein gene.

Beta-microseminoprotein (MSP) is a small protein (94 amino acids) synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. Restriction endonuclease mapping of human genomic DNA with a human MSP cDNA probe identified a 19 kilobase (Kb) hybridizing band in both EcoRI and BamHI digestions. Subsequently, the 19 Kb EcoRI fragment of human genomic DNA containing the MSP gene was isolated and cloned into an EMBL4 phage vector. Screening of the recombinant phages resulted in the isolation of one clone containing the MSP gene. Restriction endonuclease mapping and sequence analysis of this clone revealed the human MSP gene of approximately 15 Kb in length. The gene contains four exons and three large introns of approximately 6, 1, and 7 Kb.

Amino Acid Sequence↗
PubMed1989

Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium.

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the conversion of a variety of glycine-extended peptides into biologically active alpha-amidated product peptides in a reaction dependent on copper, ascorbate, and molecular oxygen. We have isolated and sequenced cDNAs representing the two major classes of PAM mRNA in the adult rat heart atrium. The two types of cDNA, rPAM-1 and rPAM-2, are identical except for the deletion of a 315-base-pair segment within the protein coding region in rPAM-2, suggesting that rPAM-1 and rPAM-2 arise by alternative splicing. Northern analysis using a cDNA probe derived from within the 315-base-pair region deleted in rPAM-2 visualized the larger of the PAM mRNAs in adult rat atrium and not the smaller, indicating that the presence or absence of this 315-nucleotide segment is a major feature distinguishing the two size forms of PAM mRNA. The 105 amino acid segment that distinguishes the two forms of atrial PAM contains a consensus N-glycosylation site and a paired basic amino acid site of potential importance in endoproteolytic processing. Comparison of the nucleotide sequences of rat, frog, and bovine PAM cDNAs reveals an extremely well conserved segment in the 3' untranslated region. The high degree of conservation in amino acid sequence throughout the catalytic, intragranular, and cytoplasmic domains of rat atrium, bovine pituitary, and frog skin PAM suggests that both the catalytic and noncatalytic domains of the protein subserve important functions.

Amino Acid Sequence↗
PubMed1988

Polypeptide fluoroacetate derivatives. Synthesis and 19F n.m.r.

The synthesis and characterization of monofluoroacetyl (MFAc) functionalized haptens are described. These were covalently bound to polypeptide carriers (bovine serum albumin and poly-D-lysine) by primary amine/succinimide ester or primary amine/acid chloride coupling. Epitopic densities of the resulting antigens were determined by both 19F n.m.r. and picryl sulfonic acid assays. 19F n.m.r. experiments defining the stability of MFAc ester and amide linkages as a function of media (including in vitro), time, and temperature are presented. These results indicate that MFAc functionalized antigens are well suited for further immunologic studies.

Fluorine↗
PubMedDate not supplied

Tryptophan hydroxylase is expressed by photoreceptors in Xenopus laevis retina.

Serotonin has important roles, both as a neurotransmitter and as a precursor for melatonin synthesis. In the vertebrate retina, the role and the localization of serotonin have been controversial. Studies examining serotonin immunoreactivity and uptake of radiolabeled serotonin have localized serotonin to inner retinal neurons, particularly populations of amacrine cells, and have proposed that these cells are the sites of serotonin synthesis. However, other reports identify other cells, such as bipolars and photoreceptors, as serotonergic neurons. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the serotonin synthetic pathway, was recently cloned from Xenopus laevis retina, providing a specific probe for localization of serotonin synthesis. Here we demonstrate that the majority of retinal mRNA encoding TPH is present in photoreceptor cells in Xenopus laevis retina. These cells also contain TPH enzyme activity. Therefore, in addition to being the site of melatonin synthesis, the photoreceptor cells also synthesize serotonin, providing a supply of the substrate needed for the production of melatonin.

Animals↗