A monoclonal antibody that detects vimentin-related proteins in invertebrates

Summary Drosophila melanogaster contains a 46 000 MW cytoplasmic protein which is immunologically related to the intermediate filament protein vimentin of vertebrates. A monoclonal antibody raised against this protein was used to study its cross-reactivity with other vertebrate and invertebrate cells. Indirect immunofluorescence showed filamentous meshworks in all species tested. Protein blotting was used to determine the molecular weights of the proteins responsible for the wide range of cross-reactivity of this antibody. We present evidence that vimentin-like proteins are also present in invertebrates and form a cytoplasmic network in Paramecium. Furthermore, we demonstrate in vertebrates and invertebrates the presence of high molecular weight polypeptides which are immunologically related to vimentin.     

A multistep bioinformatic approach detects putative regulatory elements in gene promoters

Background  

Searching for approximate patterns in large promoter sequences frequently produces an exceedingly high numbers of results. Our aim was to exploit biological knowledge for definition of a sheltered search space and of appropriate search parameters, in order to develop a method for identification of a tractable number of sequence motifs.     

A low-copy-number Sorghum DNA sequence that detects hypervariable EcoRV fragments

A sorghum genomic DNA clone that hybridized on Southern blots in simple but different patterns to fragments produced by digestion of DNA from the parents of an F₂ mapping population was hybridized to EcoRV-digested DNA from 53 accessions. Forty-six different fragment patterns were observed, each comprised of from one to ten bands. Much less variability was detected in EcoRI than EcoRV digests of a selected subset of the accessions. Base-sequence analysis of the clone did not reveal a functional identity for the sequence and the clone does not contain repeated sequences often associated with hypervariable loci. Clones such as this will be especially useful in evaluating germplasm diversity and in identifying the potential parentage of hybrids.     

Stabilization signals: a novel regulatory mechanism in the ubiquitin/proteasome system

The turnover of cellular proteins is a highly organized process that involves spatially and temporally regulated degradation by the ubiquitin/proteasome system. It is generally acknowledged that the specificity of the process is determined by constitutive or conditional protein domains, the degradation signals, that target the substrate for proteasomal degradation. In this review, we discuss a new type of regulatory domain: the stabilization signal. A model is proposed according to which protein half-lives are determined by the interplay of counteracting degradation and stabilization signals.     

Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation

A single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extension (SP-A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypeptide at a primary site (Cys(-)(1)-Asn(1)) and a secondary site (Gly(-)(4)-Ile(-)(3)). In this study, we used site-directed mutagenesis and heterologous expression in vitro and in insect cells to the examine mechanisms that may lead to alternative signal peptidase cleavage including alternative translation initiation at two in-frame AUGs (Met(-)(30) and Met(-)(20)), a suboptimal context for hydrolysis at the primary cleavage site, or cotranslational protein modifications that expose an otherwise cryptic secondary cleavage site. In vitro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa primary translation products on SDS-PAGE analysis, while translation of cDNAs encoding Met-30Ala and Met-20Ala mutations resulted in only the single 28 and 29 kDa molecular mass species, respectively. These data are consistent with translation initiation at both Met(-)(30) and Met(-)(20) during in vitro synthesis of SP-A. The Met-30Ala mutation reduced expression of the longer isoform in insect cells, indicating that the Met(-)(30) site also contributes to eucaryotic protein expression. Forcing translation initiation at Met(-)(30) by optimizing the Kozak consensus sequence surrounding that codon or by mutating the Met(-)(20) codon resulted in preferential expression of the longer SP-A isoform but reduced overall expression of the protein almost 10-fold. Both isoforms were generated to some degree whether translation was initiated at the codon for Met(-)(30) or Met(-)(20), indicating that the site of translation initiation is not the sole determinant of isoform generation and suggesting that either the context of the primary cleavage site is suboptimal or that cotranslational modifications affect cleavage. Preventing N-terminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-)(1) by substitution with serine markedly enhanced cleavage at the Gly(-)(4)-Ile(-)(3) bond, but substitution with alanine enhanced cleavage at the Cys(-)(1)-Asn(1) bond. We conclude that rat SP-A isoforms arise by a novel mechanism that includes both alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.     

A new aspect of the RNA bacteriophages translation control mechanism.

The polarity effect of the coat protein gene of the ribonucleic acid of RNA bacteriophages on the polymerase gene translation will be taken as the basis of the polymerase translation control mechanism. A further condition for this mechanism discussed in this work is the dependence of the phage RNA replication on host cell translation factors. The ribosome binding sites of the phage RNA play a decisive role to realize the control mechanism coding for definite ribosome binding probabilities. The relation between them quantifies the reached polymerase concentration in the early phase of the development of the RNA bacteriophage system in the infected cell.     

Effectiveness of distal gene translation in polycistrons depends upon the arrangement of regulatory signals on a template

The role of the translational terminator and initiator signals arrangement for two adjacent genes in polycistronic mRNA has been studied. Semisynthetic beta-galactosidase gene (lacZ) of E. coli and fragment of phage M13 DNA (with promoter PVIII, gene IX, and part of gene VIII) were used for constructing of the IX-VIII-lacZ artificial polycistronic operon. Cloning of the constructs into pBR322 vector resulted in a number of pLZ381N plasmids differing by the mutual arrangement of gene VIII translation terminator codon and SD site and initiator codon (SD-ATG-region) of lacZ gene. The mutual arrangement of gene VIII terminator codon and SDlacZ-ATG region has been altered by means of deletions and insertions that have not affected lacZ translation initiation signals. The beta-galactosidase (beta-Gal) synthesis in E. coli harbouring different types of pLZ381N plasmids has been found to depend on type of cistron coupling (gene VIII and lacZ). The overlapping of terminator and initiator codons (ATGA) for genes VIII and lacZ (type I of polycistrons) provide approximately equal translational level for both cistrons. On the other side, levels of beta-Gal synthesis in case of polycistrons type II (gene VIII stop-codon position at the beginning of SDlacZ or 10 nucleotides upstream) were 20-30 times as high as for type I. Differences in beta-Gal levels have also been found for variants of VIII-lacZ coupling in types IV and III polycistrons (the SDlacZ-ATG region in 27-50 nucleotides downstream from the proximal cistron VIII stop-codon, which, in turn, is 41 nucleotides upstream this terminator). These data cannot be explained on the basis of possible secondary structure including the SDlacZ-ATG region and other parts of polycistronic mRNA. In all these cases similarly stable stem-loop structures have been found. Therefore, the arrangement of the translation termination and initiation signals for two adjacent genes in essential for distal gene translation efficiency. One can imagine that ribosome or its 30S subpartical, stalling on the proximal gene terminator codon, affects the distal gene translation initiation.     

Investigation on the kinetic mechanism of octopine dehydrogenase. A regulatory behavior.

The kinetic scheme of octopine dehydrogenase of Pecten maximus L., a monomeric enzyme obeying a bi-ter sequential mechanism, was completed, essentially in the forward reaction, by steady-state studies over a wide range of substrate concentration at pH 7.0. Deviation from the Michaelis-Menten behavior with respect to NAD+ and other significant kinetic data led us to ascribe for octopine dehydrogenase mechanism the mnemonical enzyme concept. In addition, another regulatory behavior can be envisaged involving the formation of two dead-end complexes enzyme.NADH.D-octopine and enzyme.NAD+.pyruvate.L-arginine.     

A proposed mechanism for multiplication of neural signals

The probability of the joint occurrence of two statistically independent events is the product of the probabilities of the individual events. This fact is used to show that a neuron which detects coincident arrivals of spikes from two input neurons can function as a multiplier, i.e. its average output spike frequency is proportional to the product of the average input spike frequencies. The theoretical analysis is checked in two ways: (a) Computer simulations confirm the derived expressions for the output frequency and show that increasing the jitter in the input spike trains improves the operation of the multiplier by making the output spike train more regular (b) Experimentally recorded spike trains are used to demonstrate that the type and amount of jitter present in real spike trains is adequate for satisfactory operation of the proposed scheme for multiplication. The operating characteristics of the proposed multiplier make it an attractive candidate for the multiplicative mechanism that is involved in the optomotor response of insects.     

Rheumatoid factor blocks regulatory Fc signals

Immunosuppressed cultures of murine spleen cells, partly deprived of T cells and antigen-stimulated, can be reconstituted to near full activity in their antibody-forming cell response with murine rheumatoid factors (RF). The dose of RF required for recovery of 50% of the reconstitutable immune response was 10-100 ng and reconstitution was blocked by intact murine IgG added to the cultures. IgG subclass specificity of RF was demonstrated; RF specific for IgG2a was more potent than RF specific for IgG1 in reconstituting the response. Synergy was observed between RF added at culture initiation and late-acting B-cell differentiation factors. The greater the degree of T-cell deprivation, the more stringent the conditions needed for reconstitution. Suitable conditions for reconstitution with profound T-cell depletion included the limited reconstitution by specific RF, the synergistic action of RF with late-acting T-cell-replacing supernatants, and multiple additions of a number of RFs to the cultures on Days 0, 1, and 2. RF was also shown to block Fc-dependent immunosuppression by added antigen-antibody complexes. These results are interpreted as favoring the hypothesis put forward previously that the normal production of RF acts to reduce T-cell dependency by preventing negative Fc signal transmission by immune complexes on the B-cell surface. Abnormal production of RF may be a primary destabilizer of the immune responses leading to autoimmunity.     

A DNA marker for human chromosome 8 that detects alleles of differing sizes

A random, unique DNA sequence has been isolated and assigned to human chromosome 8. This sequence (D8MGV1) recognizes two alleles that differ in size by 700 bp.     

A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins

Asparagine-linked (N-linked) protein glycosylation has been observed in all domains of life, including most recently in bacteria and is now widely considered a universal post-translational modification. However, cell-based production of homogeneous glycoproteins for laboratory and preparative purposes remains a significant challenge due in part to the complexity of this process in vivo. To address this issue, an easily available and highly controllable Escherichia coli-based cell-free system for the production of N-linked glycoproteins was developed. The method was created by coupling existing in vitro translation systems with an N-linked glycosylation pathway reconstituted from defined components. The translation/glycosylation system yielded efficiently glycosylated target proteins at a rate of hundreds of micrograms/milliliters in half a day. This is the first time a prokaryote-based cell-free protein synthesis system has generated N-linked glycoproteins.