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PubMed1997

Sec12p requires Rer1p for sorting to coatomer (COPI)-coated vesicles and retrieval to the ER.

In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER. The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins. In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi. In contrast, rer3 mutants accumulate Sec12-derived hybrid proteins carrying early Golgi modifications. We found that rer3 mutants harbour unique alleles of the alpha-COP-encoding RET1 gene. ret1 mutants, along with other coatomer mutants, fail to retrieve KKXX-tagged type I transmembrane proteins from the Golgi back to the ER. Surprisingly rer3-11(=ret1-12) mutants do not affect this kind of ER recycling. Pulse-chase experiments using these mutants show that alpha-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins. Rer1p protein may be directly involved in the retrieval process since it also recycles between the early Golgi and ER in a coatomer (COPI)-dependent manner. Rer1p may act as an adapter coupling the recycling of non-KKXX transmembrane proteins like Sec12p to the coatomer (COPI)-mediated backward traffic.

Coatomer Protein↗
PubMed1988

Presence of neurotensin and neuromedin-N within a common precursor from a human pancreatic neuroendocrine tumor.

An acid extract of a human neuroendocrine pancreatic adenoma was found to contain very high concentrations of immunoreactive neurotensin (iNT; approximately 130 mumol/L) as well as immunoreactive neuromedin-N (iNMN; approximately 40 mumol/L), portions of which (iNT, 0.2%; iNMN, 30%) were found in large molecular forms. Processing of the large forms could be mimicked by treatment with pepsin, which increased their immunoreactivity 15- to 20-fold (iNT) and 1- to 2-fold (iNMN), liberating a peptide similar to NMN and 2 fragments of NT [primary product, NT-(4-13)]. Biochemical characterizations using gel electrophoresis, isoelectric focusing, and high pressure liquid chromatography indicated that the large forms were highly basic (pI 8.5-9.5) proteins with a mol wt of about 20K (78% of the total), 45K (8%), and 60K (4%). The 20K protein contained iNT and iNMN in a 1:1 ratio, while a slightly smaller species contained only NMN. These results are in agreement with cDNA studies of canine intestinal mRNA, indicating the presence of a 170-amino acid precursor containing 1 copy each of NT and NMN. They further indicate that within this tumor differential processing of precursor occurred, resulting in a NT to NMN ratio of about 3:1, with additional NMN stored in large molecular forms.

Adenoma↗
PubMed1996

Identification of the dipteran Leu-callatostatin peptide family: characterisation of the prohormone gene from Calliphora vomitoria and Lucilia cuprina.

The prohormone gene encoding the Leu-callatostatin peptides has been isolated from a Calliphora vomitoria genomic DNA library and its homologue was cloned from genomic and cDNA libraries of another blowfly species, Lucilia cuprina. Gene and prohormone structure and organisation are essentially identical in the two species. The Leu-callatostatin gene consists of at least 3 exons. The prohormone is encoded on exons two and three and the two blocks of putative Leu-callatostatin peptides are carried on separate exons. It is 180 amino-acids long, begins with a short signal peptide and contains two blocks of tandemly arranged Leu-callatostatin peptides separated by an acidic spacer region. The prohormone contains 5 copies of the C-terminal sequence -YX FGL characteristic of the Leu-callatostatin family. Complete endoproteolytic processing at all possible pairs of basic amino acids would generate 5 different Leu-callatostatin octapeptides. Two larger Leu-callatostatins could be released if processing was not complete at two of the sites. None of the 3 peptides encoded in the first block was identified in previous purification studies of the callatostatin peptides. The second block, located at the carboxyl end of the prohormone, contains two peptide sequences identical to the previously isolated Leu-callatostatins 1 and 4. The absence of independent copies of Leu-callatostatins 2 and 3 on the prohormone establishes that endoproteolytic cleavage of the precursor does not invariably proceed to completion and that Leu-callatostatin 2 must be derived by N-terminal processing of the parent peptide Leu-callatostatin 1. Reverse transcriptase PCR analysis of mRNA from brain and midgut, the two major sites of Leu-callatostatin expression, shows that the prohormone sequence at these two sites is identical, ruling out the possibility that different populations of peptides are expressed in these two tissues as a result of alternative RNA splicing.

Amino Acid Sequence↗
PubMed2003

Interhemispheric visuo-motor integration in humans: the effect of redundant targets.

We used event-related functional magnetic resonance imaging (fMRI) to investigate the functional locus of response facilitation during parallel visuo-motor processing. In a simple reaction-time task, subjects typically respond faster to two copies of the same stimulus than to a single copy. This facilitation, called the redundant-target effect, can occur at three functional levels: perceptual, 'cognitive' or motor. Normal right handers were studied while performing a simple reaction-time task to unilateral (left or right) and bilateral light flashes. Subjects were instructed to respond with their right index finger. Reaction times were faster to bilateral light flashes than to unilateral ones, even right flashes. Greater fMRI signal for bilateral stimuli compared to unilateral ones was observed in the left precentral and postcentral gyrus, and in the right precentral gyrus. A greater fMRI signal for bilateral and for unilateral left stimuli, compared to unilateral right stimuli, was observed in an area of the right intraparietal sulcus. These results support the hypothesis that the functional locus of response facilitation during parallel visuo-motor processing is premotor.

Adult↗
PubMed1992

Prospects for the genetic engineering of milk.

Milk and milk products comprise a substantial fraction of the protein intake of the industrialised West. The establishment of germline manipulation techniques in cows offers opportunities for directly manipulating milk composition to produce products with enhanced nutritional and processing properties. The major milk proteins are encoded by a small number of abundantly expressed single-copy genes and a number of possible manipulations are described. Milk proteins exhibit complex interactions with each other and with other constituents of milk. It will, therefore, be necessary to utilise model systems to evaluate the consequences of these proposed changes before embarking upon the costly and time-consuming process of manipulating the bovine genome.

Animals↗
PubMed1998

A stochastic model for the rapid emergence of specific vertebrate immunity incorporating horizontal transfer of systems enabling duplication and combinational diversification.

Recent molecular data indicate that the antigen-specific combinatorial immune response is restricted to jawed vertebrates where it is found in representatives of all class from cartilagenous fishes to mammals. Here, we analyse the relatively rapid emergence of the combinatorial system terms of three stochastic process, with the system reaching essentially full capacity in immunoglobulin recognition elements and diversification and recombination of gene segments in an evolutionary span of time of less than 20 million years. The mechanisms for inducibility were coopted from ancient and widely spread processes in phylogeny for regulation of cell division. The proposed process of formation entailed the evolution of unknown ancestral genes into those specifying bona fide immunoglobulin domains, and the generation of multiple copies of these via a series of events facilitated by horizontal transfer of site-specific recombinases and recombination signal sequences most probably from microbial and fungal sources. The second process is one of rapid "decay" (evolution) which occurred in about 10 million year under stringent selective conditions to generate proper conserved canonical sequences. The third process is that of the long term evolution of these characteristic immunoglobulin domains over the 450 million years since their emergence. As a first approximation the rates of these three processes were computed using first order differential equations. The rate of formation has a magnitude of 10-7 substitutions per site per year, and that of rapid modifications is 10-8 substitutions per site per year. The long term rate of immunoglobulin evolution is comparable to that of other moderately conserved proteins, (1-3) x 10-9 substitutions per site per year). This model is testable by searching for "footprints" of microbial and fungal DNA processing enzymes and recombination mechanisms. The hypothesis raises the general concept that horizontal transfer of genes facilitating rearrangement and duplication can catalyse major steps of macroevolution.

Animals↗
PubMed2005

Easy-to-prepare assembly array of Tungsten microelectrodes.

This paper provides a detailed process flow for fabricating an easy-to-prepare, inexpensive, dense array of tungsten microelectrodes. We designed the process flow to minimize routine tasks by separating an initial preparation of a master mold from a routine preparation of substrate replication, array assembly and tip processing. Sandblast processing first produced a glass mold with a pattern of a series of protruding lines at a requested interval of a few hundred micrometers. Copying the groove pattern onto polystyrene mass-produced a replica substrate. Tungsten probes were then aligned on the substrate, and the tips of probes were finely processed in the block.

Action Potentials↗
PubMed2003

The role of the Aspergillus niger furin-type protease gene in processing of fungal proproteins and fusion proteins. Evidence for alternative processing of recombinant (fusion-) proteins.

We have characterized growth and protein processing characteristics of Aspergillus niger strains carrying a disrupted allele of the previously cloned and characterized kexB gene [Appl. Environ. Microbiol. 66 (2000) 363] encoding a furin-type endoprotease. Deletion of the single-copy gene confirms it to be non-essential but disruptant strains exhibit a morphologically distinct phenotype characterized by hyperbranching. Processing of homologous pro-proteins and fusion proteins comprised of a heterologous protein fused down-stream of glucoamylase and separated at the fusion junction by an endoproteolytic cleavage site was compared in wildtype and mutant strains of A. niger. We show that maturation of the native glucoamylase requires KexB, whereas maturation of aspergillopepsin does not. The processing of fusion proteins carrying Lys-Arg requires KexB, although alternative endoproteases are capable of cleaving protein fusions at sites adjacent to Lys-Arg.

Aspergillus niger↗
PubMed1986

Fine-structure analysis of the processing and polyadenylation region of the herpes simplex virus type 1 thymidine kinase gene by using linker scanning, internal deletion, and insertion mutations.

Most eucaryotic mRNAs are polyadenylated. In higher eucaryotes, the sequence AATAAA is located 7 to 30 base pairs (bp) upstream from the site of processing and polyadenylation and is a critical part of the signal for processing and polyadenylation. Efficient cleavage and polyadenylation also require sequences downstream of polyadenylation sites. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT box (18 of 19 consecutive residues are G or T) previously shown to be required for efficient processing and polyadenylation of tk mRNA (C. N. Cole and T. P. Stacy, Mol. Cell. Biol., 5:2104-2113). To define further the sequence requirements for efficient polyadenylation, we prepared linker scanning, internal deletion, and small insertion mutations in the polyadenylation region of the tk gene. These mutations were analyzed by S1 nuclease protection analysis of cytoplasmic RNA isolated from transfected Cos-1 monkey kidney cells. When the proximal AATAAA was deleted, no tk mRNA polyadenylated in the normal region was detected, whereas replacement of the second AATAAA with an XbaI linker had no effect on polyadenylation. When various portions of the GT box were replaced with linker, the amount of tk mRNA produced was reduced to 23 to 82% of the normal amount, but polyadenylation in the normal region was never abolished. Thus, no single portion of the GT box was absolutely required. In some cases, extended transcripts, polyadenylated at a cryptic site within pBR322, were detected. A spacing of 6 bp between AATAAA and the GT box was too short for efficient processing and polyadenylation. A spacing of 30 bp appeared to work almost as efficiently as did the wild-type spacing of 18 bp. Taken together, these results indicate that efficient polyadenylation requires both AATAAA and downstream GT-rich sequences. In addition, processing and polyadenylation are affected both qualitatively and quantitatively by sequences at polyadenylation sites and at more distant locations.

Animals↗
PubMed1986

Thyrotropin-releasing hormone precursor: characterization in rat brain.

To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.

Amino Acid Sequence↗
PubMed1985

Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing, and incorporation into mature Sindbis virions.

We have obtained a vaccinia virus recombinant which contains a complete cDNA copy of the 26S RNA of Sindbis virus within the thymidine kinase gene of the vaccinia virus genome. This recombinant constitutively transcribed the Sindbis sequences throughout the infectious cycle, reflecting the dual early-late vaccinia promoter used in this construction. The Sindbis-derived transcripts were translationally active, giving rise to both precursor and mature structural proteins of Sindbis virus, including the capsid protein (C), the precursor of glycoprotein E2 (PE2), and the two mature envelope glycoproteins (E1 and E2). These are the same products translated from the 26S mRNA during Sindbis infection, and thus these proteins were apparently cleaved, glycosylated, and transported in a manner analogous to that seen during authentic Sindbis infections. By using epitope-specific antibodies, it was possible to demonstrate that recombinant-derived proteins were incorporated into Sindbis virions during coinfections with monoclonal antibody-resistant Sindbis variants. These results suggest that all the information necessary to specify the proper biogenesis of Sindbis virus structural proteins resides within the 26S sequences and that vaccinia may provide an appropriate system for using DNA molecular genetic manipulations to unravel a variety of questions pertinent to RNA virus replication.

Biological Transport↗
PubMed2005

Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress.

Mitochondrial biogenesis and mitochondrial DNA (mtDNA) maintenance depend on coordinated expression of genes in the nucleus and mitochondria. A variety of intracellular and extracellular signals transmitted by hormones and second messengers have to be integrated to provide mammalian cells with a suitable abundance of mitochondria and mtDNA to meet their energy demand. It has been proposed that reactive oxygen species (ROS) and free radicals generated from respiratory chain are involved in the signaling from mitochondria to the nucleus. Increased oxidative stress may contribute to alterations in the abundance of mitochondria as well as the copy number and integrity of mtDNA in human cells in pathological conditions and in aging process. Within a certain level, ROS may induce stress responses by altering expression of specific nuclear genes to uphold the energy metabolism to rescue the cell. Once beyond the threshold, ROS may cause oxidative damage to mtDNA and other components of the affected cells and to elicit apoptosis by induction of mitochondrial membrane permeability transition and release of pro-apoptotic proteins such as cytochrome c. On the basis of recent findings gathered from this and other laboratories, we review the alterations in the abundance of mitochondria and mtDNA copy number of mammalian cells in response to oxidative stress and the signaling pathways that are involved.

Animals↗
PubMed1989

Tissue-specific expression of the alternately processed Drosophila myosin heavy-chain messenger RNAs.

The myosin heavy-chain (Mhc) gene of Drosophila is a single-copy gene from which four messenger RNAs are transcribed. Two of these mRNAs, CA-1 and CA-2, are expressed in all stages of development when Mhc mRNA is detected. The 3' ends of these mRNAs differ by alternate choice of poly(A) addition sites. Two additional Mhc mRNAs, CBA-1 and CBA-2, are detected only in midpupal to adult stages of development. The 3' ends of these mRNAs are alternately polyadenylated as the above mRNAs; however, these mRNAs contain an additional alternately spliced exon. We have used in situ hybridization to tissue sections to determine the tissue-specific expression of the alternately processed Mhc mRNAs. Four probes were used in the in situ hybridization experiments: one that detects all Mhc mRNAs, one that is specific for mRNA molecules polyadenylated at the downstream site 2, one that is specific for alternately spliced mRNAs containing the B exon, and one that is specific for Mhc mRNAs Ca-1 and CA-2. This last probe is an oligodeoxynucleotide, while the others are single-stranded RNA molecules synthesized in vitro. Our results demonstrate that the alternate splicing of Mhc mRNAs is muscle-cell-type-specific during pupal development, while the polydenylation site usage at the downstream site 2 is not muscle-cell-type-specific during either embryonic or pupal development.

Animals↗
PubMed1984

Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system.

Plasmids are autonomously replicating DNA molecules that are present in defined copy numbers in bacteria. This number may for some plasmids be very low (2-5 per average cell). In order to be stably inherited, replication and partitioning of the plasmid have to be strictly controlled. Plasmids carry genetic information for both processes. In the present paper we summarize what is known about the replication control system of one low-copy-number plasmid, R1, belonging to the FII incompatibility group. We do so because the FII group seems to be one of the best understood examples with respect to genetics, molecular biology, and physiology of the replication control system. The paper is not a classical review, but rather an essay in which we discuss the aspects of replication control that we regard as being important.

Bacterial Proteins↗
PubMed1997

The role of host factors in the population dynamics of selfish transposable elements.

Previous models of the evolution of selfish transposable genetic elements have failed to include the possibility that transposition may be limited by shortage of a host-encoded factor. The titration of host factors may be important in limiting the rate of transpositional increase in these elements. This will be exacerbated if multiple copies of the host factor protein must bind simultaneously to the target element. In the case of the Drosophila melanogaster P transposable element, which can exist as autonomous and as non-autonomous copies, there is evidence that a host-encoded protein, IRBP, is required for the transposition process. We have produced a specific model of the invasion of a host population by the P element, in which we have incorporated the requirement for the multiple binding of a host factor. We find that, for the P family, in which it is apparently transposition itself that creates selective harm to the host, the effect of selection in the context of host factor limitation is to drive up copy number. This can result in a novel high copy number-low transposition state. We also find that host factor limitation reinforces the tendency for transposable elements that create sterility to be replaced by their deletion derivatives.

Animals↗
PubMed1988

Anatomical connections of the prepositus and abducens nuclei in the squirrel monkey.

The primary goal of this investigation was to identify the areas of the brainstem and cerebellum that provide afferent projections to the nucleus prepositus hypoglossi in primates. After horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) was injected into the prepositus in squirrel monkeys (Saimiri sciureus), the largest populations of retrogradely labeled neurons were found in the vestibular nuclei, the contralateral perihypoglossal nuclei, and the medullary and pontine reticular formation. Unlike the cat, the prepositus in Saimiri received substantial projections from the nucleus raphe dorsalis and the central mesencephalic reticular formation, whereas few or no labeled cells were found in the cerebellar cortex, the superior colliculus, or the nucleus reticularis tegmenti pontis. By comparing the afferents to the prepositus with those to the abducens nucleus, we found that all regions projecting to the abducens also projected to the prepositus, without exception. Anterogradely transported WGA-HRP showed that the major brainstem recipients of prepositus efferents were the vestibular and perihypoglossal nuclei, the inferior olive, the medullary reticular formation, and the extraocular motor nuclei. In the cerebellar cortex, the prepositus projected to restricted regions of crura I and II as well as the caudal vermis and vestibulocerebellum. The many parts of the oculomotor system receiving input from the prepositus and the parallel innervation of the prepositus and the abducens by a large number of premotor centers lend support to the hypothesis that the prepositus may distribute an efference copy of motor activity, and may also play an important role in the process of neural integration.

Abducens Nerve↗
PubMed1988

Molecular and cellular characterization of drug resistant hamster cell lines with alterations in ribonucleotide reductase.

Ribonucleotide reductase consists of 2 protein components frequently called M1 and M2. Hydroxyurea specifically inhibits DNA synthesis by interacting with the M2 protein and destroying a unique tyrosyl-free radical. We have carried out a molecular and cellular characterization of 2 Chinese hamster ovary cell lines exhibiting either low (HN(R)-AT) or relatively high (H(R)-R2T) resistance to the cytotoxic effects of hydroxyurea. Both drug-resistant lines have an increased level of ribonucleotide reductase activity. EPR measurements for tyrosyl-free radical content and studies with M1-specific antibodies indicated that the elevation in enzyme activity was entirely due to an increase in the M2 component. Studies with M1 cDNA showed that both drug-resistant cell lines contained a wild-type level of M1 mRNA and a wild-type M1 gene copy number. Studies with M2 cDNA indicated that the 2 drug-resistant lines possessed elevated levels of M2 message that could explain the observed increase in M2 component. The elevation of M2 mRNA in the most resistant line, H(R)-R2T, was due to an increase in M2 gene copy number. The low resistant cell line, HN(R)-AT, exhibited a wild-type M2 gene copy number, indicating that the increase in M2 gene message occurred through a process other than gene amplification. Enzyme kinetic studies with partially purified preparations from both drug resistant lines showed reduced sensitivity to hydroxyurea and to the negative allosteric effector, dATP. In addition to hydroxyurea, H(R)-R2T cells were also resistant to several other drugs whose site of action is the M2 component. Furthermore, H(R)-R2T cells were not cross-resistant to colchicine or puromycin, suggesting that hydroxyurea-resistant cells do not share the multi-drug resistance phenotype, which is frequently associated with cross-resistance to these drugs.

Animals↗
PubMed1991

Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences.

Antigenic diversity is generated in the wall-less pathogen Mycoplasma hyorhinis by combinatorial expression and phase variation of multiple, size-variant membrane surface lipoproteins (Vlps). The unusual structural basis for Vlp variation was revealed in a cluster of related but divergent vlp genes, vlpA, vlpB and vlpC, which occur as single chromosomal copies. These encode conserved N-terminal domains for membrane insertion and lipoprotein processing, but divergent external domains undergoing size variation by loss or gain of repetitive intragenic coding sequences while retaining a motif with distinctive charge distribution. Genetic analysis of phenotypically switched isogenic lineages representing ON or OFF expression states of Vlp products ruled out chromosomal rearrangement or frameshift mutations as mechanisms for Vlp phase variation. However, highly conserved vlp promoter regions contain a tract of contiguous A residues immediately upstream of the -10 box which is subject to frequent mutations altering its length in exact correspondence with the ON and OFF phase states of specific genes. This suggests a mechanism of transcriptional control regulating high frequency phase variation and random combinatorial expression of Vlps. The multiple levels of diversity embodied in the vlp gene cluster represents a novel adaptive capability particularly suited for this class of wall-less microbe.

Amino Acid Sequence↗