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PubMed2000

HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human.

HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2beta and HE2gamma proteins have a 25-amino acid deletion near the C-terminus, and HE2gamma isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the beta1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The beta2 form is expressed in principal cells primarily in efferent ducts.

3' Untranslated Regions↗

PubMed1999

A sequence in the Escherichia coli fdhF "selenocysteine insertion Sequence" (SECIS) operates in the absence of selenium.

The UGA codon context of the Escherichia coli fdhF mRNA includes an element called the selenocysteine insertion sequence (SECIS) that is responsible for the UGA-directed incorporation of the amino acid selenocysteine into a protein. Here, we describe an extended fdhF SECIS that includes the information for an additional function: the prevention of UGA readthrough under conditions of selenium deficiency. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively, and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Here, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium, the SECIS enables one of two alternatives for the translational machinery: either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.

Arginine↗

PubMed1980

Biosynthesis of parathyroid hormone.

Figure 11 summarizes our present concepts of the biosynthetic sequence for parathyroid hormone, deduced largely from observations in vitro. Many aspects of the presumed process whereby preproparathyroid hormone is converted via proparathyroid hormone to the hormone remain unclarified and require much further study, as is true for many other prehormones. These studies, however, coupled with (1) further investigations of intracellular degradation of parathyroid hormone, if this indeed operates in vivo; (2) the proteolytic conversion of secreted hormone in peripheral tissues; and (3) analysis of transcriptional control of biosynthesis of parathyroid hormone, using radioactive cDNA for hybridization studies of mRNA production and turnover, hold great promise for further understanding of critical regulatory factors central to expression of the actions of parathyroid hormone.

Adenylyl Cyclases↗

PubMed1992

Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

Base Sequence↗

PubMed1994

Primary structure and functional properties of an epithelial K channel.

Expression cloning in Xenopus oocytes was used to identify a clone for a renal K channel. The clone, named ROMK2, was obtained from a cDNA library constructed in the plasmid vector pSPORT using size-selected poly(A)+ RNA from whole rat kidney. ROMK2 consists of 1,837 nucleotides, with an open reading frame of 1,116 bases predicted to code for a 372-amino acid peptide. The clone appears to be a splice variant of a recently reported K channel (ROMK1) from rat renal outer medulla (Ho, K.H., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. Nature Lond. 362: 31-37, 1993). Northern blot analysis indicates that ROMK2 is expressed in renal cortex, medulla, and papilla. Expression in other tissues appears to be much lower. The functional properties of the channel as measured in Xenopus oocytes indicate its close relationship to ROMK1 and more distant relationship to the inward rectifier K channel (IRK1) (Kubo, Y, T.J. Baldwin, Y. N. Jan, and L. Y. Jan. Nature Lond. 362: 127-133, 1993). The inward conductance of the channel is a saturable function of external K, with a half-maximal conductance at <5 mM. The selectivity sequence for ion permeability based on reversal potential measurements was K > Rb > NH4 > Na, Li. The conductance to Rb was only one-half that to K. Extracellular Ba2+ and Cs+ blocked the channel in a voltage-dependent manner. The high sensitivity of Cs+ block to voltage is consistent with the channel's operating as a multi-ion pore. The channel was blocked by high concentrations (100 microM) of glibenclamide. It did not appear to be blocked by extracellular Na+ or tetraethyl-ammonium ion. Patch-clamp measurements indicated a single-channel conductance of 30 pS in the presence of 110 mM K and high open probability that was weakly dependent on voltage. This channel may be involved in maintaining the membrane potential of renal cells and/or mediating renal K secretion.

Amino Acid Sequence↗

PubMed1983

Synthesis of two polypeptide subunits of an aminoacyl tRNA synthetase as a single polypeptide chain.

E. coli aminoacyl tRNA synthetases are typically comprised of a single type of polypeptide chain. Glycine tRNA synthetase is an exception, and is comprised of two different subunits. Previous work showed that glyS encodes both subunits in a tandem arrangement of coding regions which are in the same reading frame. Nine nucleotides separate the TAA stop of the first coding segment (alpha-subunit) from the ATG start of the second one (beta-subunit). A plasmid containing glyS was put into four different ochre suppressor strains. In three of them, significant quantities of an alpha-beta fusion protein were synthesized in maxicells, in genetic backgrounds which retained cellular proteases. This shows that the fusion protein is stable in vivo and suggests that Gly-tRNA synthetase is operationally a single polypeptide which is the ancestor of the two subunits.

Amino Acid Sequence↗

PubMed1990

Expression of a ciliate gene in Escherichia coli using a suppressor tRNA to read the UAA and UAG glutamine codons.

Most ciliates use a particular genetic code where the standard stop codons UAA and UAG encode glutamine. Ciliate genes cannot therefore be expressed in heterologous systems such as Escherichia coli. To overcome this problem, we worked out a system of inducible suppression to permit efficient readthrough of UAAs and UAGs: a strong UAA tRNA suppressor that inserts glutamic acid was cloned downstream from a tac promoter whose efficiency was reduced by a transcription terminator. This system proved to be operational (1) to suppress UAG mutations by wobble pairing in an E. coli lacI-lacZ gene fusion and (2) to read through at least eight UAA glutamine codons in a Paramecium alpha-tubulin gene, as detected by Western blotting and colony hybridization. This work opens the way for cloning Ciliate genes from expression libraries and for expressing particular sequences without extended in vitro mutagenesis. A similar approach can be envisaged for expression of genes from Mycoplasma, mitochondria or other genomes that use non-standard genetic codes.

Animals↗

PubMed1988

A mammalian viral enhancer confers transcriptional regulation in yeast.

Transcriptional enhancers are DNA sequences that regulate RNA transcription from linked promoters by binding to cellular proteins (trans-activators). In the mammalian virus SV40, initiation of transcription is controlled, in part, by a strong 72-base pair enhancer. We show that yeast cells contain a factor that binds specifically to a key DNA motif in the SV40 enhancer, the P element that is essential for viral transcription in mammalian cells. The P element shows sequence similarity to a yeast DNA transcriptional regulatory element, GCN4, that controls transcription of genes that code for amino acid biosynthetic enzymes. Insertion of the SV40 enhancer or single or multiple copies of the P element itself upstream from the cytochrome Cyc-1 promoter places the hybrid viral-yeast transcription unit under metabolic control in yeast cells. These studies suggest that the SV40 P element and its complementary trans-activator represent a conserved transcriptional control mechanism that operates on widely divergent functions in evolution.

Base Sequence↗

PubMed1983

Repression of glycoprotein synthesis and release of surface coat during transformation of Trypanosoma brucei.

The biosynthesis of the variant surface glycoprotein (VSG) and its release from the surface of Trypanosoma brucei 427 variant clone MITat 1.4 (117) during in vitro transformation of bloodstream trypomastigotes to procyclic trypomastigotes was investigated. After transfer to the transformation medium at 27 degrees C, VSG synthesis is repressed with a half-time, t1/2 = 30 min. Concomitantly VSG-specific mRNA is lost suggesting that repression operates at the transcriptional level. The expression-linked extra gene copy, which codes for VSG, is retained during and after completion of transformation. After repression of VSG synthesis, surface VSG is shed from the cells into the culture medium. During release part of VSG (apparent mol. wt. 61 000) is proteolytically cleaved to a product (apparent mol. wt. 51 000) which represents the N-terminal domain of the protein as judged by the absence of the carbohydrate moiety normally linked to the C terminus.

Animals↗

PubMed1989

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid.

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.

Bacillus↗

PubMed1990

A family of expression vectors based on the rrnB P2 promoter of Escherichia coli.

We describe here the construction of a family of expression vectors, based on the P2 promoter of the Escherichia coli rrnB gene by removing regulatory sequences downstream of the Pribnow-box and replacing them with the lac operator. These vectors allow cloning of foreign genes in such a way that their products are synthesized either in the form of fusion proteins of different length, or without fusion partners, with or without the original translational initiation signals. One of the vectors contains a synthetic oligothreonine-coding sequence that helps to stabilize the product of the cloned gene. These vectors allow high-level regulated expression of foreign genes, even if their products are relatively short peptides.

Amino Acid Sequence↗

PubMed1981

The sequence of human serum albumin cDNA and its expression in E. coli.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.

Amino Acid Sequence↗

PubMed1999

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Amino Acid Sequence↗

PubMed1997

Induction of rabbit cyclooxygenase 2 in the anterior uvea following glaucoma filtration surgery.

PURPOSE: This study was undertaken to evaluate for the presence of cyclooxygenase 2 (COX2) gene expression in the anterior uvea of rabbits following glaucoma filtration surgery. METHODS: One of the following surgical procedures were performed on the right eye of New Zealand white albino rabbits: (1) paracentesis (2.5 mm limbal incision); (2) iridectomy through a 2.5 mm limbal incision; (3) lamellar scleral flap formation or (4) full glaucoma filtration surgery. The animals were sacrificed within 3 hours of post-surgery, and the anterior uveal tissues were isolated. Polymerase chain reaction-based techniques were employed to assay for the presence of COX2 transcript. RESULTS: A partial coding sequence of the previously unreported rabbit COX2 gene was obtained. COX2 mRNA was detected in the operated eyes of animals that underwent either full filtration surgery or iridectomy through a limbal incision. CONCLUSIONS: In normal rabbit anterior uveal tissue, there appears to be minimal expression of COX2 message. After experimental glaucoma filtration surgery, there is rapid induction of COX2 message.

Amino Acid Sequence↗

PubMed1993

Decreased accumulation of beta 1-adrenergic receptor, G alpha s and total myosin heavy chain messenger RNAs in the left ventricle of senescent rat heart.

The expression of genes coding for the beta 1-adrenergic receptor (beta 1-AR), the alpha subunit of Gs and total myosin heavy chain (MHC) was compared between left ventricles (LV's) from young (6-7 weeks old) and old (22 months old) rats. The mRNA levels were quantitated by Northern or Slot blots analyses using specific DNA probes. Ageing was found to be associated with a reduction in beta 1-AR (77%), G alpha s (33%) and, total MHC (51%) mRNA levels with no concomitant change in 18S RNA and poly(A+) mRNA levels. These results indicate that transcriptional and/or post-transcriptional mechanisms participate in the control of beta-adrenergic receptor density during ageing. As in the senescent LV, beta 1-AR mRNA level is reduced in the hypertrophied LV, whereas the level of G alpha s mRNA is reduced in the senescent but not in the hypertrophied LV. From our data we conclude (1) that a dual mechanism may operate during ageing, mechanical factors indirectly regulating beta 1-AR mRNA level, while changes in G alpha s mRNA level do not depend on hemodynamic load and (2) that the re-expression of beta-MHC mRNA does not compensate for the decreased accumulation of alpha-MHC mRNA which results in a large decrease in the level of total MHC mRNA in the senescent LV.

Aging↗

PubMed1990

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis.

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.

Animals↗

PubMed1997

Conservation of the myoglobin gene among Antarctic notothenioid fishes.

We determined the myoglobin cDNA sequence for seven Antarctic notothenioid fish species. These data identify mutations in the myoglobin gene for Champsocephalus gunnari and Pagetopsis macropterus, two icefish species that lack detectable quantities of the polypeptide but express myoglobin mRNA. a third species lacking myoglobin polypeptide, Chaenocephalus aceratus, is devoid of myoglobin mRNA and accordingly failed to produce myoglobin products on polymerase chain reaction (PCR) amplification. Myoglobin cDNA sequences were highly conserved among the species the express the protein, particularly in the coding region. Sequence variation among the myoglobin-expressing channichthyid species was 2.0% to 2.9% in the coding region and 2.6% to 3.3% over the entire cDNA. The same extent of variation, 1.6% to 3.2% in the coding sequence and 2.8% to 3.7% overall, was observed between the icefishes and more distantly related, red-blooded nototheniid species. The two species expressing mutant myoglobin mRNA, C. gunnari and P. macropterus, exhibited the highest degree of sequence variation among the fish myoglobins examined. Drift in the myoglobin sequence in these two species, and conservation of myoglobin cDNA among fishes from two distinct families, suggest that a selective pressure operates to maintain myoglobin in the species that express the protein.

Amino Acid Sequence↗

PubMed1995

Triplet code-independent programming of living systems organisation by DNA: the link with intelligence and memory.

Previous suggestions from this laboratory (3), (a) that within its molecular electronic structure, DNA houses a computer-analog program of immense complexity, operating independently of, but complementary to, triplet coding and (b) that, inter alia, this program is the driving force for organising and executing the construction of species individuals in three dimensions, are extended in the present communication. It is now concluded that the DNA program also embodies an 'intelligence' component, which extends its organising ability both qualitatively and quantitatively beyond any of the heavily circumscribed 'self-organising' attributes claimed to be associated with naturally occurring inanimate systems. Further, that as part of the developmental process, a program component organises the fabrication of mammalian central nervous systems, including that of human beings with the associated attributes of intelligence, creativity and constructional skills. It is further suggested that the sophisticated random access memory system associated with human beings in particular may be explicable in terms of an extension of the DNA programming system: basically this involves the latter operating as computer-type 'hardware' for the storage of long-term memory and interacting with, primarily, glial cell RNA, acting as 'software' and storing short term traces. Finally, it is suggested that such an interrelationship between DNA/RNA molecular electronic structures can provide the necessary memory storage capacity and flexibility and also facilitates random access to the long-term DNA memory store.

Animals↗