Heterologous Regions Research Articles

Molecular Characterisation of Equine Herpesvirus 1 Isolates from Cases of Abortion, Respiratory and Neurological Disease in Ireland between 1990 and 2017.

Multiple locus typing based on sequencing heterologous regions in 26 open reading frames (ORFs) of equine herpesvirus 1 (EHV-1) strains Ab4 and V592 was used to characterise 272 EHV-1 isolates from 238 outbreaks of abortion, respiratory or neurological disease over a 28-year period. The analysis grouped the 272 viruses into at least 10 of the 13 unique long region (UL) clades previously recognised. Viruses from the same outbreak had identical multi-locus profiles. Sequencing of the ORF68 region of EHV-1 isolates from 222 outbreaks established a divergence into seven groups and network analysis demonstrated that Irish genotypes were not geographically restricted but clustered with viruses from all over the world. Multi-locus analysis proved a more comprehensive method of strain typing than ORF68 sequencing. It was demonstrated that when interpreted in combination with epidemiological data, this type of analysis has a potential role in tracking virus between premises and therefore in the implementation of targeted control measures. Viruses from 31 of 238 outbreaks analysed had the proposed ORF30 G2254/D752 neuropathogenic marker. There was a statistically significant association between viruses of the G2254/D752 genotype and both neurological disease and hypervirulence as defined by outbreaks involving multiple abortion or neurological cases. The association of neurological disease in those with the G2254/D752 genotype was estimated as 27 times greater than in those with the A2254/N752 genotype.

Exploring the Role of ATP Hydrolysis in the RecA Protein via Stalled DNA Intermediates

A central participant in the recombinational DNA‐repair system is the recombinase protein, RecA, which acts as a DNA pairing enzyme that is responsible for promoting an exchange of homologous DNA strands. Physical studies have revealed three DNA binding sites buried within the interior of the RecA filament. This current understanding of RecA is incomplete as it fails to explain how a four‐strand DNA exchange occurs between two duplex DNA fragments.The bacterial RecA homologue is unique in that it couples ATP‐hydrolysis with DNA strand exchange. ATP hydrolysis occurrs uniformly throughout the RecA filament, with only a small fraction of ATP used for filament disassembly. The mechanism by which the RecA filament utilizes the rest of ATP hydrolysis to perform DNA strand exchange reactions is not fully understood. My aim is to explore the hypothesis that the majority of ATP utilized by the reaction is hydrolyzed at binding sites located longitudinally along both the interior and exterior of the filament, providing the energy needed to rotate one segment of DNA around another.This will be explored by performing RecA mediated strand‐exchange reactions with a variety of linear, double stranded DNA substrates containing heterologous regions. When the RecA protein is run into a heterologous barrier, a stalled DNA‐protein intermediate molecule is the product. Formation of these intermediates has been obtained. Imaging with Electron Microscopy shows topological changes that offer fundamental insight into the universal mechanics of RecA‐mediated strand exchanges. Furthermore, evidence shows that introduction of the protein Uup modifies the structure of the intermediates, providing insight into Uup's function.

Protective antigen and extractable antigen 1 based chimeric protein confers protection against Bacillus anthracis in mouse model

Recombinant bivalent chimeric protein was generated comprising of domain 4 of protective antigen (PA4) and carboxy terminal region of extractable antigen 1 (EA1C) by overlap extension PCR. The immunogenicity and protective efficacy of recombinant chimeric protein (PE) and protein mixture (PAEA) along with the individual components, PA4 and EA1C were evaluated in this study. We found that PE and PAEA exhibited higher endpoint titer and elevated IgG1 response. Compared to PA4 and EA1C, the chimeric protein PE and protein mixture PAEA exhibited 1.52 and 1.39 times more proliferative effect on lymphocytes in vitro. The spore uptake by anti-PE and anti-PAEA antibodies was significantly more than the individual components. We further evaluated the effects of antisera on the toxins in vitro and in vivo. Anti-PE and anti-PAEA antibodies displayed nearly 80% protection against crude toxin activity on RAW 264.7 cell lines. We further demonstrated that the anti-PE and anti-PAEA antibodies displayed better protection in controlling the edema induced by crude toxin. Passive immunization with anti-PE and anti-PAEA provided protection against toxin challenge in mice. The present study reveals that the chimeric protein consisting of heterologous regions of PA and EA1 can render better protection than PA4 or EA1C alone against toxins and bacilli.

Functional reorganization of brain networks in patients with painful chronic pancreatitis

The underlying pain mechanisms of chronic pancreatitis (CP) are incompletely understood, but recent research points to involvement of pathological central nervous system processing involving pain-relevant brain areas. We investigated the organization and connectivity of brain networks involved in nociceptive processing in patients with painful CP. Contact heat-evoked potentials (CHEPs) were recorded in 15 patients with CP and in 15 healthy volunteers. The upper abdominal area (sharing spinal innervation with the pancreatic gland) was used as a proxy of 'pancreatic stimulation', while stimulation of a heterologous region remote to the pancreas (right forearm) was used as a control. Subjective pain scores were assessed by visual analogue scale. The brain source organization and connectivity of CHEPs components were analysed. After pancreatic area stimulation, brain source analysis revealed abnormalities in the cingulate/operculo-insular network. A posterior shift of the operculo-insular source (p = 0.004) and an anterior shift of the cingulate source (p < 0.001) were seen in CP patients, along with a decreased strength of the cingulate source (p = 0.01). The operculo-insular shift was positively correlated with the severity of patient clinical pain score (r = 0.61; p = 0.03). No differences in CHEPs characteristics or source localizations were seen following stimulation of the right forearm. CP patients showed abnormal cerebral processing after stimulation of the upper abdominal area. These changes correlated to the severity of pain the patient was experiencing. Since the upper abdominal area shares spinal innervation with the pancreatic gland, these findings likely reflect maladaptive neuroplastic changes, which are characteristic of CP.

Programmed Protection of Foreign DNA from Restriction Allows Pathogenicity Island Exchange during Pneumococcal Transformation

In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of S. pneumoniae through programmed protection and incorporation of foreign DNA.

Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination.

Error-prone ZW pairing and no evidence for meiotic sex chromosome inactivation in the chicken germ line.

In the male mouse the X and Y chromosomes pair and recombine within the small pseudoautosomal region. Genes located on the unsynapsed segments of the X and Y are transcriptionally silenced at pachytene by Meiotic Sex Chromosome Inactivation (MSCI). The degree to which MSCI is conserved in other vertebrates is currently unclear. In the female chicken the ZW bivalent is thought to undergo a transient phase of full synapsis at pachytene, starting from the homologous ends and spreading through the heterologous regions. It has been proposed that the repair of the ZW DNA double-strand breaks (DSBs) is postponed until diplotene and that the ZW bivalent is subject to MSCI, which is independent of its synaptic status. Here we present a distinct model of meiotic pairing and silencing of the ZW pair during chicken oogenesis. We show that, in most oocytes, DNA DSB foci on the ZW are resolved by the end of pachytene and that the ZW desynapses in broad synchrony with the autosomes. We unexpectedly find that ZW pairing is highly error prone, with many oocytes failing to engage in ZW synapsis and crossover formation. Oocytes with unsynapsed Z and W chromosomes nevertheless progress to the diplotene stage, suggesting that a checkpoint does not operate during pachytene in the chicken germ line. Using a combination of epigenetic profiling and RNA–FISH analysis, we find no evidence for MSCI, associated with neither the asynaptic ZW, as described in mammals, nor the synaptic ZW. The lack of conservation of MSCI in the chicken reopens the debate about the evolution of MSCI and its driving forces.

Reliability of Static and Dynamic Quantitative Sensory Testing in Patients With Painful Chronic Pancreatitis

Quantitative sensory testing (QST) has proven to be an important instrument to characterize mechanisms underlying somatic and neuropathic pain disorders. However, its reliability has not previously been established in patients with visceral pain. We investigated the test-retest reliability of static and dynamic QST in patients with visceral pain due to chronic pancreatitis. Sixty-two patients (38 men, 53 [11] y) with painful chronic pancreatitis were included. Static QST comprised sensory thresholds for pressure and electrical stimulation performed in the ventral and dorsal T10 dermatomes (sharing spinal innervation with the pancreas, ie, pancreatic viscerotomes) and in 4 heterologous regions (control areas). Dynamic QST comprised conditioned pain modulation. Measurements were obtained from 2 subsequent test sessions separated by 1 week. The reliability of static QST was generally high, with the best test-retest performance seen for pressure pain thresholds (intraclass correlation coefficients [ICC], 0.74) and electrical sensation thresholds (ICC, 0.66). In contrast, the reliability of dynamic QST was poor (ICC, 0.01). For static QST measures, the reliability was higher for pain thresholds compared with suprapain thresholds (P < 0.01). No differences between assessments in the pancreatic viscerotomes compared with heterologous regions were seen (P = 0.6). Sensory thresholds in the pancreatic viscerotomes and control areas were reproducible over time. In contrast, dynamic QST measurements reflecting active central modulation of pain processing state (ie, conditioned pain modulation) were not stable over time and showed considerable variability. These factors should be taken into consideration in case QST is used to follow disease mechanisms, drug effects, or effects of pain intervention.

IntDOT Interactions with Core- and Arm-Type Sites of the Conjugative Transposon CTnDOT

CTnDOT is a Bacteroides conjugative transposon (CTn) that has facilitated the spread of antibiotic resistances among bacteria in the human gut in recent years. Although the integrase encoded by CTnDOT (IntDOT) carries the C-terminal set of conserved amino acids that is characteristic of the tyrosine family of recombinases, the reaction it catalyzes involves a novel step that creates a short region of heterology at the joined ends of the element during recombination. Also, in contrast to tyrosine recombinases, IntDOT catalyzes a reaction that is not site specific. To determine what types of contacts IntDOT makes with the DNA during excision and integration, we first developed an agarose gel-based assay for CTnDOT recombination, which facilitated the purification of the native IntDOT protein. The partially purified IntDOT was then used for DNase I footprinting analysis of the integration site attDOT and the excision sites attL and attR. Our results indicate that CTnDOT has five or six arm sites that are likely to be involved in forming higher-order nucleoprotein complexes necessary for synapsis. In addition, there are four core sites that flank the sites of strand exchange during recombination. Thus, despite the fact that the reaction catalyzed by IntDOT appears to be different from that typically catalyzed by tyrosine recombinases, the protein-DNA interactions required for higher-order structures and recombination appear to be similar.

Rad52-mediated DNA annealing after Rad51-mediated DNA strand exchange promotes second ssDNA capture

Rad51, Rad52, and RPA play central roles in homologous DNA recombination. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 has two distinct activities: to recruit Rad51 onto single-strand (ss)DNA that is complexed with the ssDNA-binding protein, RPA, and to anneal complementary ssDNA complexed with RPA. Here, we report that Rad52 promotes annealing of the ssDNA strand that is displaced by DNA strand exchange by Rad51 and RPA, to a second ssDNA strand. An RPA that is recombination-deficient (RPA(rfa1-t11)) failed to support annealing, explaining its in vivo phenotype. Escherichia coli RecO and SSB proteins, which are functional homologues of Rad52 and RPA, also facilitated the same reaction, demonstrating its conserved nature. We also demonstrate that the two activities of Rad52, recruiting Rad51 and annealing DNA, are coordinated in DNA strand exchange and second ssDNA capture.

Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

The mechanisms of RecA-mediated three-strand homologous recombination are investigated at the single-molecule level, using magnetic tweezers. Probing the mechanical response of DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of the progression of the exchange in real time, both from the point of view of the RecA-bound single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show that strand exchange is able to generate torsion even along a molecule with freely rotating ends. RecA readily depolymerizes during the reaction, a process presenting numerous advantages for the cell's 'protein economy' and for the management of topological constraints. Invasion of an untwisted dsDNA by a nucleoprotein filament leads to an exchanged duplex that remains topologically linked to the exchanged single strand, suggesting multiple initiations of strand exchange on the same molecule. Overall, our results seem to support several important assumptions of the monomer redistribution model.

DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA

Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.

Identificação de Pectobacterium carotovorum subsp. brasiliensis através de PCR-RFLP do gene recA

Pectobacterium carotovorum subsp. brasiliensis foi proposta como o principal agente causal da canela preta da batata (Solanum tuberosum) no Brasil. Com o objetivo de identificar essa subespécie, oligonucleotídeos iniciadores foram selecionados a partir de regiões heterólogas do gene recA existentes entre P. carotovorum subsp. brasiliensis ATCC BAA-41 e outras pectobactérias disponíveis no GenBank e P. carotovorum subsp. carotovorum BAB1. No entanto, os oligonucleotídeos iniciadores apresentaram baixa especificidade. O produto da PCR do gene recA, um fragmento de ± 730 pb, de 38 estirpes de P. chrysanthemi e das diferentes subespécies de P. carotovorum, foi digerido com as endonucleases de restrição TasI e HhaI. Estas enzimas foram selecionadas com base na seqüência do gene recA das estirpes P. carotovorum subsp. brasiliensis ATCC BAA-416 (581 pb) e P. carotovorum subsp. carotovorum BAB1 (626 pb). A análise do PCR-RFLP com as enzimas TasI e HhaI gerou sete e 12 padrões, respectivamente. A combinação dos resultados permitiu a separação em 13 grupos distintos e a discriminação de P. carotovorum subsp. brasiliensis.

A Tolerance of DNA Heterology in the Mammalian Targeted Gene Repair Reaction

Targeted gene repair consists of at least two major steps, the pairing of an oligonucleotide to a site bearing DNA sequence complementarity followed by a nucleotide exchange reaction directed by the oligonucleotide. In this study, oligonucleotides with different structures were designed to target a stably integrated (mutant) enhanced green fluorescent protein (EGFP) gene and used to direct the repair of a single base mutation. We show that the efficiency of correction is influenced by the degree of DNA sequence homology existing between the oligonucleotide and target gene. Correction is reduced when a heterologous stretch of DNA sequence is placed in the center of the oligonucleotide and the mismatched base pair is then formed near the terminus. The negative impact of heterology is dependent on the type of DNA sequence inserted and on the size of the heterologous region. If the heterologous sequence is palindromic and adopts a secondary structure, the negative impact on the correction frequency is removed, and wild-type levels of repair are restored. Although differences in the efficiency of correction are observed in various cell types, the effect of structural changes on gene repair is consistent. These results reveal the existence of a directional-specific repair pathway that relies on the pairing stability of a bilateral complex and emphasize the importance of sequence homology between pairing partners for efficient catalysis of gene repair.

In vitro analysis of sequence requirements for the excision reaction of the Bacteroides conjugative transposon, CTnDOT

CTnDOT, a Bacteroides conjugative transposon (CTn), initiates its transfer by excising to form a circular intermediate. This process has been shown to be complex, involving an unusual DNA intermediate with a short region of heterology and several CTn-encoded proteins. No information was available, however, about the sizes or sequence requirements of the att sites (attL and attR) at the ends of the integrated element where the processing occurs during excision. Using a newly developed in vitro competition excision assay, we have now localized attL to 153 bp and attR to 179 bp. Excision of CTnDOT involves staggered cuts that produce 5 bp chromosomal sequences at either end of the CTn. These 5 bp sequences (coupling sequences) form a region of heterology in the excised circular intermediate. Site-directed mutations that made the coupling sequences complementary and removed the region of heterology had no effect on excision. Thus, heterology is not essential. Mutagenesis of sequences adjacent to the coupling sequences revealed a 6 bp site in attR that was essential for excision. Mutating the analogous region in attL had little effect on excision. Regions within the attL site that appear to play a role in excision were found by introducing small insertions (phasing mutations) that could interfere with protein-protein or protein-DNA interactions. Similar insertion mutations in attR had no significant effect on excision. These results support the hypothesis that the CTnDOT excision reaction is asymmetrical with respect to likely protein binding sites and involves multiple protein-DNA interactions.

Differential Detection of Five Mouse-Infecting Helicobacter Species by Multiplex PCR

Several species of helicobacter have been isolated from laboratory mice, including H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, which appear to be the most common. The most widely used published method for molecular detection of these agents is PCR amplification of a conserved region of 16S rRNA, but differential speciation requires restriction enzyme digestion of the amplicons. This study was undertaken to determine PCR conditions that would simultaneously and specifically identify each of the five common species without restriction enzyme analyses. First, we designed novel and specific PCR primers for H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, using sequences from the heterologous regions of 16S rRNA. Because of comigration of amplified products, we next identified P17, an H. bilis-specific protein; P25, an H. hepaticus-specific protein; and P30, an H. muridarum-specific protein by screening genomic DNA expression libraries of each species. Primers were designed from these three genes, plus newly designed, species-specific 16S rRNA primers for H. rodentium and H. typhlonius that could be utilized for a five-plex PCR. The sizes of the amplicons from H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius were 435, 705, 807, 191, and 122 bp, respectively, allowing simultaneous detection and effective discrimination among species.

RuvAB-directed branch migration of individual Holliday junctions is impeded by sequence heterology.

The Holliday junction, the key intermediate of recombination, is generated by strand exchange resulting in a covalent connection between two recombining DNA molecules. Translocation of a Holliday junction along DNA, or branch migration, progressively exchanges one DNA strand for another and determines the amount of information that is transferred between two recombining partners. In Escherichia coli, the RuvAB protein complex promotes rapid and unidirectional branch migration of Holliday junctions. We have studied translocation of Holliday junctions using a quantitative biochemical system together with a 'single-molecule' branch migration assay. We demonstrate that RuvAB translocates the junctions through identical DNA sequences in a processive manner with a broad distribution of individual branch migration rates. However, when the complex encounters short heterologous sequences, translocation of the Holliday junctions is impeded. We conclude that translocation of the junctions through a sequence heterology occurs with a probability of bypass being determined both by the length of the heterologous region and the lifetime of the stalled RuvAB complex.

Mechanism of DI RNA Formation in Tombusviruses: Dissecting the Requirement for Primer Extension by the Tombusvirus RNA-Dependent RNA Polymerase in Vitro

Tombusviruses, which are positive-strand RNA viruses of plants, frequently generate defective interfering (DI) RNAs that consist of three to four noncontiguous segments of the parental RNA. Replicase jumping was postulated to cause multiple deletions leading to the de novo formation of DI RNAs in planta. This model was tested using a partially purified RNA-dependent RNA polymerase (RdRp) preparation from tombusvirus-infected plants in vitro. The tombusvirus RdRp was capable of primer extension without the need for sequence complementarity between the primer and the acceptor template in vitro, although the most efficient primer extension was obtained with primers forming a 5-bp duplex with the acceptor region. Primers forming 14- to 20-bp duplexes with the acceptor region were used less efficiently by the tombusvirus RdRp in vitro. In addition, primers with 3' noncomplementary nucleotides were also extended by the tombusvirus RdRp, albeit with a reduced efficiency. The preference of the tombusvirus RdRp for short base-paired primers in vitro is consistent with the lack of extended sequence similarities at the junction sites in the de novo generated tombusvirus-associated DI RNAs. The in vitro experiments also revealed that the acceptor region plays a significant role in primer extension. Comparison of tombusvirus-derived, heterologous and artificial acceptor regions revealed that the conserved regions present in DI RNAs are the best acceptor regions when they are available in the minus-strand orientation. These data suggest that recombination/deletion events may be more frequent at some regions, rather than occurring randomly throughout the parental genome. In addition, these findings support a model that predicts a higher frequency of replicase jumping, i.e., recombination/deletion events, during plus-strand synthesis than during minus-strand synthesis.

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1)

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12–p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis λgt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5′-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5′-end. Escherichia coli transfected with the construct containing the alternative 5′-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5′-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5′-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.

Overproduction of the Opi1 repressor inhibits transcriptional activation of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae.

Transcription of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae is repressed by high concentrations of inositol and choline. The ICRE (inositol/choline-responsive element), which is necessary and sufficient for regulation by phospholipid precursors, functions as a binding site for the heterodimeric Ino2/Ino4 activator. ICRE-dependent transcription becomes constitutive in the absence of the Opi1 repressor. Opi1 contains a leucine zipper motif and two glutamine-rich stretches. In this work we describe a molecular analysis of OPI1 function and expression. Opi1 mutant variants altered at the leucine zipper and a glutamine-rich region, respectively, were no longer functional repressors. In contrast, an Opi1 deletion variant lacking the N-terminal 106 amino acids still mediated negative regulation. Although the leucine zipper suggests that Opi1 may act as a DNA-binding protein, our data do not support a direct interaction with the ICRE. Despite its function as an antagonist of INO2 and INO4, expression of OPI1 is stimulated by an upstream ICRE. Overexpression of OPI1 under control of the GAL1 promoter severely inhibited activation of ICRE-dependent genes, leading to inositol-requiring cells. Growth inhibition of GAL1-OPI1 was observed with INO2 and INO4 alleles activated by either the natural promoter or a heterologous control region. Although induction of GAL1-OPI1 strongly repressed ICRE-dependent gene expression, the concentration of the Ino2/Ino4 activator remained unchanged. This finding suggests that differential expression of phospholipid biosynthetic genes may occur even in the presence of a constant amount of the specific activator.