lacZ Sequences Research Articles

LacZ sequences prevent regulated expression of housekeeping genes

In order to dissect the MHC class I H-2K gene regulatory sequences, we previously generated transgenic mice containing various H-2K/lacZ fusion genes. However, contrary to transgenes where H-2K sequences were fused to other coding sequences, none of the lacZ fusion transgenes was widely expressed like H-2K gene. We now show that this silencing also occurs when lacZ is inserted into a larger H-2K genomic construct including promotor and other regulatory elements. Because the 5′ H-2K region contains a CpG island, we suspected that the presence of lacZ coding sequences was interfering with the mechanism by which the H-2K promoter region is normally unmethylated and transcriptionally active. Indeed, we show that in high (>10) copy number transgenic mice, insertion of lacZ sequences in the vicinity of the H-2K promoter results in partial or complete methylation of the H-2K CpG island. However, in low (1–3) copy number transgenic mice no methylation was observed but the transgene was still silent, suggesting that the silencing effect of lacZ does not only rely on abnormal CpG methylation. Intriguingly, when the H-2/lacZ construct was introduced via embryonic stem (ES) cells, regulated transgene expression was observed in several chimaeric embryos derived from independent ES clones, but never in adult chimaeras. Combined with the fact that, despite much effort, it has been very difficult to generate ‘blue’ mice, our results highlight the transcription-silencing effect of lacZ sequences when they are associated with regulatory sequences of ubiquitously expressed genes.

Deletion errors generated during replication of CAG repeats.

Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer. Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases. To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes. Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions. DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats. Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides. Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction. Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative. This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches.

SOS-dependent A→G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA

Gas chromatography/isotope dilution–mass spectrometry with selected ion monitoring (GC/IDMS–SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C→A (∼20-fold increase in SOS-conditions), G→A (9-fold increase) and G→C (4.5-fold increase). Very few G→T transitions were found. A particularly large group of A→G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A→G transitions in SOS-induced bacteria.

Effects of homology length and donor vector arrangement on the efficiency of double-strand break-mediated recombination in human cells.

We use an Epstein-Barr virus (EBV) plasmid model chromosome system to study how different donor plasmid constructs affect recombination stimulated by an I-SceI-induced double-strand break in the target sequence in human cells. The entire 3.5 kb lacZ gene was efficiently recombined into a target EBV vector lacking lacZ sequences, but having limited homology to the donor plasmid. A donor plasmid with lacZ flanked by sequence homologous to the target consistently generated gene conversion events and was more effective than a donor carrying lacZ outside the same sequence homology. Reducing the length of homology between the target and donor from 5.5 kb to 1 kb caused only a 3-fold drop in recombination frequency, contrasting with the exponential dependence on homology length seen when no DSB is present in the target. These results document a DSB-induced 175-fold increase in recombination of a heterologous gene into a target, requiring only limited flanking homology.

Cre-loxP System Confers Cell Lineage-Specific Expression of a Reporter Gene in Murine Preimplantation Development.

The Cre-loxP site-specific recombination system is useful for tracing the fate of cells via transient expression of the Cre recombinase gene directly introduced into the cells with a vector carrying loxP sites. In this study, we used this system to examine whether cell lineage-specific expression of a reporter gene occurs in murine preimplantation embryos. An expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken β-actin promoter (CAG), a portion of the rabbit β-globin gene (including the 2nd intron, 3rd exon and 3' noncoding region), loxP-flanked DNA sequence [containing enhanced green fluorescent protein (EGFP) cDNA and chloramphenicol acetyltransferase gene (CAT)], and lacZ gene encoding E. coli β-galactosidase (β-gal), was constructed. When pCETZ-17 DNA was microinjected into the pronuclei of fertilized eggs, 30.0% (6/20) of the surviving 2-cell embryos exhibited distinct fluorescence in both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for β-gal. When both plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, all (12/12) embryos exhibited low or no fluorescence, but 66.7% (8/12) exhibited positive staining for β-gal activity in one or both blastomeres. This indicates that transient expression of Cre removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only 2-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing 2-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 2 days up to 8-cell stage. When the developing 8-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for β-gal activity was observed in all (7/7) embryos. These findings indicate that this Cre-loxP system, which is based on transient expression of the Cre gene directly introduced into nuclei of embryonic cells by microinjection, is useful for tracing cell lineage as well as for expressing a gene of interest in a lineage-specific manner in early embryogenesis

PBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli.

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.

Retroviral Transfer of Antisense Integrin α6 or α8 Sequences Results in Laminar Redistribution or Clonal Cell Death in Developing Brain

To assess the roles of two integrin alpha subunits (alpha6 and alpha8) in the developing chicken optic tectum, progenitors were infected with retroviral vectors that contained the marker gene lacZ plus antisense sequences from either the alpha6 or alpha8 integrin subunit cDNAs. On embryonic day 3 (E3), the vector was injected into tectal ventricles of chicken embryos. On E6, E7.5, E9, or later, chicken embryos were killed, and optic tecta were dissected and processed for histochemical detection of lacZ-positive cells. The antisense-bearing cell clones (descendants of a single infected progenitor) were analyzed for proliferation and migration patterns and were compared with lacZ-only vector-infected control clones. At E6, both alpha6 and alpha8 integrin antisense-containing cell clones were similar to controls. At E7.5, integrin alpha8 antisense-containing clones exhibited a cell number reduction in upper laminae (intermediate zone and tectal plate), and at E9, they exhibited a reduction in the ventricular zone as well. Integrin alpha6 antisense-containing cell clones exhibited no difference in total cell number at E9 but had a net laminar redistribution of more cells in the ventricular zone and less cells in the tectal plate. Our data show that different integrins play different roles during brain development: alpha6 integrin is essential for migration of tectal cells into specific laminae, and alpha8 integrin is essential for the survival of optic tectum cells. Also alpha8 integrin-substrate interactions may suppress early programmed cell death in premigratory and migratory neuroblasts.

A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptional elongation-associated recombination.

We have identified two novel yeast genes, THO1 and THO2, that partially suppress the transcription defects of hpr1Delta mutants by overexpression. We show by in vivo transcriptional and recombinational analysis of tho2Delta cells that THO2 plays a role in RNA polymerase II (RNA pol II)-dependent transcription and is required for the stability of DNA repeats, as previously shown for HPR1. The tho2Delta mutation reduces the transcriptional efficiency of yeast DNA sequences down to 25% of the wild-type levels and abolishes transcription of the lacZ sequence. In addition, tho2Delta causes a strong increase in the frequency of recombination between direct repeats (>2000-fold above wild-type levels). Some DNA repeats cannot even be maintained in the cell. This hyper-recombination phenotype is dependent on transcription and is not observed in DNA repeats that are not transcribed. The higher the impairment of transcription caused by tho2Delta, the higher the frequency of recombination of a particular DNA region. The tho2Delta mutation also increases the frequency of plasmid loss. Our work not only identifies a novel yeast gene, THO2, with similar function to HPR1, but also provides new evidence for transcriptional blocks as a source of recombination. We propose that there is a set of proteins including Hpr1p and Tho2p, in the absence of which RNA pol II transcription is stalled or blocked, causing genetic instability.

Retroviral transfer of herpes simplex virus-thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector

In this study we describe a new retroviral vector utilizing an internal ribosome entry site (IRES) from encephalomyocarditis virus to co-express two genes. One is the herpes simplex virus type 1 thymidine kinase gene (HSV-TK) which induces sensitivity to ganciclovir, and the second is the bacterial beta-galactosidase gene ( LacZ) which was revealed by an histochemical staining with 5-bromo-4-chloro-3-indolyl-β- d-galactopyranoside (X-Gal). We enginereed the U937 human cell line to co-express both genes and monitored transduced cells using X-Gal staining. Several transduced clones were selected. The clones exhibiting X-Gal positive cells were sensitive to ganciclovir treatment (1 μg/ml) while X-Gal negative clones were not. Monoclonal cell lines showed a single copy of the provirus integrated in their genome with the TK-IRES- LacZ sequence stably inserted in all clones. The band distribution pattern of the proviral DNA differed only at the long terminal repeat (LTR) level. Northern blot analysis of an X-Gal positive/ganciclovir sensitive clone showed an mRNA band of 6 kb with both LacZ and TK probes. An X-Gal negative/ganciclovir resistant clone was negative with both probes. This report shows: (1) a therapeutic gene can be linked to a marker gene by an IRES element achieving equivalent expression of both proteins; (2) the co-expression of a marker gene makes fluorescein-di-β- d-galactopyranoside staining possible, and consequently separation of cells expressing the LacZ gene by fluorescence activated cell sorting. Thus the cells expressing the HSV-Tk gene are enriched; (3) the use of a marker gene such as LacZ could open up interesting perspectives in gene therapy protocols because of the opportunity to monitor the transduced cells using a simple cytochemical stain.

Function of posterior HoxD genes in the morphogenesis of the anal sphincter.

Vertebrate 5'-located HoxD genes are expressed in the most caudal part of the digestive tract and their potential functions during gut development have been assessed by gene disruptions. We have inserted reporter lacZ sequences within the Hoxd-12 gene and analysed the morphology of the gut in these mice as well as in Hoxd-13 mutant animals. When homozygous, both mutations induce an important disorganization of the anorectal region. In particular, severe alterations of the smooth muscle layers of the rectum led to defective morphogenesis of the internal anal sphincter. Similarly, Hoxd-12 and Hoxd-13 functionally overlap during digit development. The function of these genes in the morphogenesis of the digestive system as well as their functional evolution are discussed.

Retinoic acid and methylation cis-regulatory elements control the mouse tissue non-specific alkaline phosphatase gene expression

To understand the mechanisms regulating the tissue non-specific alkaline phosphatase (TNAP) activity during development, we characterized cis-transcriptional regulatory elements. In embryonic cells and tissues, TNAP expression was driven preferentially by the exon 1A (E1A) promoter, one of the two promoters previously defined. Transcriptional activity of E1A promoter was up-regulated by retinoic acid (RA) through a putative RA-responsive element. Transgenic mice analysis with lacZ reporter constructs revealed negative regulatory elements within 8.5 kb of E1A promoter. Promoter sequences of endogenous TNAP in non-expressing tissues and those carried by the 8.5 kb- lacZ transgene were found to be highly methylated. A 1 kb fragment of E1A promoter increased the methylation level of lacZ and promoter sequences. The role of RA and DNA methylation in defining the embryonic expression pattern of TNAP is discussed.

Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa.

The Neurospora crassa arg-2 transcript contains an upstream open reading frame (uORF) specifying a 24-residue leader peptide and is subject to a novel form of negative translational regulation in response to arginine. The role of the arg-2 uORF in arginine-specific negative regulation was investigated by using translational fusions of wild-type and mutant arg-2 sequences to the Escherichia coli lacZ reporter gene specifying beta-galactosidase. The wild-type uORF conferred Arg-specific regulation on the reporter gene in N. crassa, but mutated or truncated uORFs did not, as determined by measurements of beta-galactosidase activity produced in N. crassa strains expressing arg-2-lacZ fusion genes. All effects on reporter gene expression were posttranscriptional, as determined by measurement of RNA levels. Both sequence-dependent and sequence-independent effects of uORFs were observed. Genes containing the wild-type uORF or a 21-codon mutated uORF showed reduced translation in comparison with that of a gene lacking a uORF. Both uORF-containing transcripts showed reduced association with polysomes relative to transcripts lacking a uORF, but only the transcript with the wild-type uORF showed a reduced average number of ribosomes associated with it in response to arginine addition. Direct translational fusions between uORF sequences and lacZ sequences indicated that the uORF is translated. Overlapping the uORF with the lacZ initiation codon indicated that ribosome reinitiation at a downstream start codon is not integral to uORF-mediated, Arg-specific translational regulation. These studies provide direct biochemical evidence for arg-2 uORF function in translational control.

Smooth Muscle α-Actin Downregulation in Cultured Chick Aortic Smooth Muscle and Neural Crest Cells Is Associated with Altered Cell Shape

A modified CXL retrovirus was used to clone an antisense smooth muscle α-actin ribozyme sequence adjacent to the reporter lacZ sequence. The virus was applied to downregulate α-actin expression in cultured smooth muscle cells obtained from chicken aortic arch and cultured neural crest cells. After infection with the ribozyme-containing CXL retrovirus both the smooth muscle and neural crest cells showed β-galactosidase activity accompanied by a reduction of smooth muscle α-actin-positive fibers. Double staining of β-galactosidase and smooth muscle α-actin using immunohistochemistry revealed that single cells infected with the CXL/ribozyme showed little to no smooth muscle α-actin protein. The absence of smooth muscle α-actin was associated with a distinct change in cellular morphology of the cultured cells, suggesting that expression of smooth muscle α-actin in cultured neural crest cells may be associated with cytoskeletal elements rather than vascular smooth muscle phenotype.

Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae.

HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with > 80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching.

Human biliverdin IXalpha reductase is a zinc-metalloprotein. Characterization of purified and Escherichia coli expressed enzymes.

Biliverdin IXalpha reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin. BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity. A cDNA clone encoding human BVR was isolated from a gamma library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA. This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed. The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein. The cDNA hybridized with a single message of approximately 1.2 kb in human kidney poly(A)-rich RNA, and appeared, by Southern blot analysis, to be the product of a single-copy gene. Sequence analysis indicated that the human reductase shows approximately 83% identity, at both the nucleotide and amino acid levels, with rat BVR. In some regions including the carboxyl terminus, protein sequence identity drops to 45%. Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat). The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions. Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio. That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent, activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity.

The roles of starvation and selective substrates in the emergence of araB-lacZ fusion clones.

The araB-lacZ fusion system has been a key case in the 'directed mutation' controversy. Fusions did not occur detectably during normal growth but formed readily after prolonged incubation on selective Ara-Lac medium. To distinguish the roles of starvation stress and selective substrates in coding sequence fusions, we applied sib selection and PCR technologies. Sib selection of the prefusion strain, MCS2, starved under aerobic conditions permitted us to isolate active fusion clones which had never been in contact with arabinose or lactose. Hence, a directive role for selective substrates is not essential. Aerobiosis was necessary for fusions to appear in glucose-starved cultures. The difference in fusion formation between normal and starved conditions is best explained by the response of a signal transduction network to physiological stimuli to activate Mu prophage joining of araB and lacZ sequences. PCR analysis revealed that direct plating on selective Ara-Lac agar yielded mostly a single class of 'standard' fusions, while sib selection yielded a broader spectrum of fusion structures. Standard fusions were found to occur within a narrow 9 bp window in lacZ. The high frequency of standard fusions in glucose-starved cultures suggested efficient and/or specific Mu action.

Identification of a potentially important antigen of pasteurella haemolytica

To identify antigens which may be important for stimulating immunity to pneumonic pasteurellosis, a bovine antiserum to whole P. haemolytica was used to screen a recombinant lambda gt11/ P. haemolytica expression library. One of the recombinant bacteriophage clones identified with the bovine antiserum, SW20C, expressed a fusion protein which was also recognized by rabbit antiserum to partially purified P. haemolytica culture supernatant and was found to be immunogenic in guinea pigs. The guinea pig antibody recognized a 100 kDa protein in P. haemolytica cell lysates. Sequence analysis of the cloned DNA from SW20C identified a fragment of 1443 bp with a small open reading frame that was contiguous with the lacZ sequence. The 153 bp P. haemolytica-specific open reading frame encoded a polypeptide of approximately 6kDa. Homology searches of Genbank and the EMBL data bases revealed no homology of this open reading frame with any other bacterial sequences including P. haemolytica leukotoxin and Ssa1. Evaluation of sera from calves that were scored either susceptible or resistant to experimental pneumonic pasteurellosis demonstrated a significant ( P < 0.001) correlation between the intensity of the antibody response to the SW20C antigen and resistance to disease.

Studies of fowlpox virus recombination in the generation of recombinant vaccines

A p7.5/β-galactosidase (7.5 lacZ) gene construct, cloned adjacent to the fowlpox virus (FPV) thymidine kinase ( tk) gene was used as a marker to identify the products of recombination as ‘blue’ FPV plaques. The rFPVs were detected as early as 4 h after the introduction of plasmid DNAs and by 72 h post-infection (p.i.) for one transfer vector comprised 0.48% of the viral population. The proportion of rFPV increased linearly from 0.073% to 0.62% as the cumulative length of homologous sequences in the transfer vector increased from 0.73 to 4.5 kb. Two approaches using a second reporter gene, the Newcastle disease virus haemagglutinin-neuraminidase (NDV HN) gene were tested to differentiate between single and double cross-over events. In one, the HN gene was cloned into the FPV tk gene and the 7.5 lacZ cloned outside of the homologous region. Progeny of a single cross-over with FPV DNA generated an unstable blue plaque containing the HN gene and subsequent intramolecular recombination resulted in excision of the 7.5 lacZ and the generation of a stable ‘white’ plaque. For virus grown in CEF cells (tk +) in the presence of 5-bromo-deoxyuridine, only those viruses which contained a tk gene disrupted by the HN gene formed plaques. This approach allowed us to easily identify rFPV containing the HN gene but lacking 7.5 lacZ or other bacterial sequences. In a second approach, a double cross-over between rFPV DNA containing a stably expressed β-galactosidase gene cloned into the tk gene (blue plaque) and plasmid DNA containing the HN gene flanked by tk sequences would allow transplacement of the 7.5 lacZ gene with the HN gene, and generating a white plaque. We were unable to generate recombinant viruses with the HN gene and which generated a white plaque, indicating that double cross-over events do not occur at a sufficiently high frequency in FPV.

Molecular cloning and characterization of prolyl endopeptidase from human T cells.

The prolyl endopeptidase (PEP) gene of human T cells was amplified by the PCR method and cloned in Escherichia coli. The complete gene consisted of 2,130 nucleotides corresponding to 710 amino acid residues with a calculated molecular mass of 80,750. The nucleotide sequence of this clone revealed that T cell PEP DNA is 48, 50, and 91% homologous to those of Flavobacterium meningosepticum, Aeromonas hydrophila, and porcine brain PEP, respectively. This gene was fused to the lacZ sequence from E. coli and expressed as a fused protein in E. coli. This fused protein exhibited PEP activity, which was inhibited by Z-Pro-prolinal, a specific inhibitor of PEP. The fused protein was purified on a beta-galactosidase specific affinity column. A polyclonal antibody was raised against the purified protein. Immunological characterization suggested that this protein is different from cytosol-soluble PEP.

Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain.

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.