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PubMed2005

An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

BACKGROUND: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process. RESULTS: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family. CONCLUSION: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

Bacterial Proteins↗

PubMed2001

A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein.

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.

Base Sequence↗

PubMed2005

[A study on polymerase chain reaction site-directed mutagenesis of prepro-parathyroid hormone in vitro].

OBJECTIVE: To study PCR site-directed mutagenesis of prepro-parathyroid hormone gene in vitro and let furin convert it into mature parathyroid hormone in human cells. METHOD: Prepro-parathyroid hormone cDNA of SD rat was cloned from its genomic gene and mutated by overlap mutant PCR, introducing furin consensus sequences (Arg-Lys-Lys-Arg). An expression pcDNA3.1/mPTH vector encoding a genetically modified prepro-parathyroid hormone cDNA was generated, and transduced to 293 cells by lipofectin-mediated DNA transfection. Forty-eight and 72 h after the transfection, the culture media were collected for further assay with radioimmunoassay. RESULTS: A fragment of prepro-parathyroid hormone gene was cloned and one site were mutated simultaneouly. After screening and sequencing of pcDNA3.1/mPTH vectors, a correctly mutated vectors was obtained. While measuring parathyroid hormone in the medium of the expressing 293 cells by RIA method, the results of transient expression were 28.34 - 52.64 pg/2.0 x 10(6)/cells/Day, which were much higher than that in control cells. CONCLUSIONS: A correctly mutated prepro-parathyroid hormone cDNA was obtained successfully, transfected, and expressed efficiently in human cells.

Animals↗

PubMed2006

New high fidelity polymerases from Thermococcus species.

Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. Phylogenetic analysis of SSU rDNA showed that the two strains were not closely related, as confirmed by an examination of the DNA polymerase sequences. Inteinless versions of each gene were generated by overlap-extension PCR and transferred into plasmid expression vectors. The proteins were produced in an Escherichia coli strain with additional copies of tRNAs corresponding to rarely used codons and purified by standard chromatographic procedures. Both enzymes were able to support PCR, but the Thermococcus 'GT' polymerase required higher concentrations of template than the enzyme from T. zilligii. Both enzymes showed 3' to 5' exonuclease activity, which was abolished in the case of T. zilligii by mutating the aspartic acid at position 141 and the glutamic acid at position 143 to alanine. Both enzymes showed a significant increase in fidelity of replication compared to the family A Thermus aquaticus DNA polymerase, in agreement with other results reported for family B polymerases with proof-reading ability.

Amino Acid Sequence↗

PubMed2005

[Construction and expression of single chain variable fragments (ScFv) against human CD19 antigen].

The genes encoding for the light and heavy chain variable regions were cloned by RT-PCR from a murine monoclonal hybridoma cell line, which could produce monoclonal antibody to recognize CD19 antigen on human B lymphocyte. Then fused the light and heavy chain variable regions together by a short peptide linker containing 15 amino acid (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant anti-CD19- ScFv was subcloned into the expression vector pET28a and induced to be expressed by IPTG in E. coli BL21. SDS-PAGE and Western blot analysis showed that the recombinant anti-CD19-ScFv gene was expressed in E. coli BL21. ScFv expression was in the form of an inclusion bodies and the purified fusion protein was obtained after a series of purification steps including cell break, inclusion body solubilization, Ni2+ metal affinity chromatography and protein refolding. Flow cytometry analysis showed that the ScFv can react with human CD19 antigen. In conclusion, recombinant anti-CD19-ScFv gene has been successful constructed and expressed in E. coli BL21, which could provide a basic study for the future target therapy to the B lymphoid leukemia and B lymphoma.

Antibodies, Monoclonal↗

PubMed2004

[Construction and expression of single chain Fv gene against domain II of human VEGF receptor II].

The genes encoding for the light and heavy chain variable regions (V(H) and V(L)) has been cloned by RT-PCR from a murine hybridoma that produced monoclonal antibody (mAb) Ycom1D3, which was against domain III of human vascular endothelial growth factor receptor II (KDRIII) and were then connected to each other by a short peptide linker containing 15 amino acids (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant Ycom1D3-ScFv gene was cloned into the expression vector pAYZ and induced to express in E. coli 16C9. SDS-PAGE and Western blot analysis showed that the recombinant Ycom1D3-ScFv gene was expressed in E. coli 16C9 and the relative molecular weight of the fusion protein is 30kD which was consistent with the theoretically predicted value. ScFv expression was in the form of an inclusion body and the purified fusion protein was obtained after a series of purification steps including cell breakage, inclusion body solubilization, TALON metal affinity chromatography and protein refolding. Flow cytometric analysis showed that the ScFv fragment can react with human umbilical vein endothelial cells (HUVECs) which express KDR on the cell surface. In Conclusion, Recombinant Ycom1D3-ScFv gene has been successfully constructed and expressed in E. coli 16C9, which could be useful in both diagnostic and therapeutic applications.

Animals↗

PubMed1995

Infectious proviral clones of chimpanzee foamy virus (SFVcpz) generated by long PCR reveal close functional relatedness to human foamy virus.

Infectious proviral clones of simian foamy virus isolated from chimpanzee (SFVcpz) were generated by long PCR. Two overlapping fragments representing the complete provirus were amplified from genomic DNA of infected cells. Four 8.8-kbp amplimers extending from base 1 of the provirus into the env gene and five 4.45-kbp amplimers reaching from env to the end of the 3'-LTR were cloned into pCR II. Subsequently, the proviral fragments were combined in a chessboard manner to generate 20 plasmids containing full-length proviral DNA. Four plasmids produced infectious virus after transfection of susceptible cells. A distinct proviral form bearing a deletion in the transactivator gene joining both exons of a second regulatory gene present in wild-type foamy virus-infected cells started to emerge 48 hr after transfection of BHK cells with infectious SFVcpz DNA. This observation supports a novel hypothesis to explain establishment of foamy virus latency. The transactivator protein Taf of SFVcpz transcomplemented for the homologous protein Bel-1 of the unique human foamy virus isolate (HFV) and Bel-1 exhibited the reciprocal activity, suggesting that HFV could represent a variant of chimpanzee foamy virus.

Animals↗

PubMed2000

Rapid-cycle PCR method to detect Bordetella pertussis that fulfills all consensus recommendations for use of PCR in diagnosis of pertussis.

No standardized PCR method is available for the laboratory diagnosis of the pertussis syndrome. Consensus recommendations for the use of PCR in the diagnosis of Bordetella pertussis infections have been proposed, and the aim of this study was to develop a method that fulfills all of these criteria. A rapid-cycle shared-primer PCR method with a microwell format and probe hybridization detection step (POR) was developed using novel oligonucleotides targeted to the outer membrane porin gene (Bordetella spp.). In specimens positive for Bordetella spp., B. pertussis was differentiated from Bordetella parapertussis and Bordetella bronchiseptica by hybridization with organism-specific oligonucleotide probes. An internal control was developed using overlap extension PCR and mouse beta-actin DNA. The analytical specificity was 100%. The analytical sensitivity was comparable to that of nested IS481 and IS1001 PCR ( approximately 1 organism per reaction). The clinical sensitivity and specificity were ascertained using 705 specimens (from 705 patients). The results were compared to those of a nested-PCR method targeting the insertion sequences IS481 and IS1001. Fifty-one specimens were positive for B. pertussis by POR and IS481 PCR. Two specimens which fulfilled a clinical definition of pertussis were positive by POR and negative by IS481 PCR. A total of 652 specimens were negative by both methods. B. parapertussis was not detected in any specimens. PCR inhibition was detected in 21 out of 705 specimens (2.98%). Thus, a rapid (4 h, including specimen preparation) PCR method which fulfills all of the consensus recommendations was developed and validated for the detection of B. pertussis.

Animals↗

PubMed1996

False-positive human immunodeficiency virus type 1 western blot tests in noninfected blood donors.

BACKGROUND: The manufacturers' criteria for a positive human immunodeficiency virus type 1 (HIV-1) Western blot (WB) test were recently revised to require reactivity to only two of the following bands: p24, gp41, and gp120/160. In a recent report, low-risk blood donors were identified in whom nonspecific reactivity to multiple env antigens in WB testing resulted in apparently false-positive WBs by these criteria. The present study was conducted to verify the existence of false-positive WBs among noninfected donors and to assess the extent of this problem. STUDY DESIGN AND METHODS: Four donors classified as WB-positive on the basis of env-only (3 cases) or p24/env-only (1 case) patterns were investigated. Index and/or follow-up specimens were tested by polymerase chain reaction (PCR), by overlapping recombinant env antigens and synthetic peptides in enzyme immunoassays, and by deglycosylated and denatured antigen WBs. WB records from American Red Cross blood centers were reviewed to determine the frequency of env-only and p24/env-only patterns, relative to all positive WBs, from 1988 through 1993. RESULTS: The four index-case donors denied risk and had stable WB reactivity during follow-up. HIV PCR was negative in all. Env reactivity was restricted to nonglycosylated gp41 epitopes; no gp120-specific reactivity was detected. For three of the four donors, env reactivity was mapped to a 20-amino acid N-terminal epitope of gp41. The rate of detecting WBs with these false-positive patterns increased from 0.6 percent of all positive WBs from 1988 to 1990 (4/776) to 8 percent in 1991 and 1992 (52/683), and then it declined to 6 percent in 1992 and 1993 (47/783). Env-only patterns predominated in 1991 and 1992, whereas p24/env-only patterns were more frequent following implementation of combined anti-HIV-1/HIV type 2 enzyme immunoassays in 1992. CONCLUSION: Low-risk blood donors can have false-positive results on WB tests. Increased detection of env-only and p24/env-only WBs appears related to the enhanced sensitivity of newer enzyme immunoassays to gp41 and p24 antibodies. Donors with these patterns should undergo follow-up testing to document the presence or absence of HIV infection.

Adult↗

PubMed2002

Identification of the calcium channel alpha 1E (Ca(v)2.3) isoform expressed in atrial myocytes.

Antisense oligonucleotides targeting the calcium channel alpha 1E (Ca(v)2.3) subunit significantly inhibit the insulin-like growth factor-1 (IGF-1)-stimulated increase in low voltage-activated (LVA) (T-type) calcium current in cultured rat atrial myocytes [Proc. Natl. Acad. Sci. U.S.A. 94(1997) 14936]. As part of a continuing effort to understand the regulation of LVA current expression in the heart, we have identified the specific alpha 1E isoform that is expressed in atrial tissue. Through reverse transcription-polymerase chain reaction (RT-PCR), nine overlapping partial clones spanning the entire coding region of the cardiac alpha 1E mRNA were obtained. The predominate isoform in atrial tissue was identified and found to be highly homologous to the alpha 1E isoform previously isolated from kidney and the islets of Langerhans [Eur. J. Biochem. 257(1998) 274]. The expression of alpha 1E in the heart occurs specifically in cardiac myocytes and not in smooth muscle or fibroblasts as demonstrated by RT-PCR performed on isolated atrial myocytes and by in situ hybridization.

Animals↗

PubMed2004

Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii.

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL(P)), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL(P)-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.

Animals↗

PubMed2005

Human galectin-2: expression profiling by RT-PCR/immunohistochemistry and its introduction as a histochemical tool for ligand localization.

Sugar-encoded information of glyco-conjugates is translated into cellular responses by endogenous lectins. Galectins stand out against other lectin families due to their wide range of functions including cell adhesion, tissue invasion or growth regulation exerted at extracellular, membrane, cytoplasmic and nuclear sites. This remarkable versatility warrants close scrutiny of their emerging network, in this study with focus on homodimeric human galectin-2. We first detected presence of specific mRNA in various tissue types by processing post mortem and surgical specimens by RT-PCR protocols. Overlap of gene expression was noted with proto-type galectins-1 and -7 and also family members from the other two subgroups. To monitor expression on the level of protein a polyclonal anti-galectin-2 antibody was raised. Immunopositivity was semi-quantitatively assessed in sections of 209 human samples establishing an array both of normal tissues and samples with inflammation or benign/malignant growth. In general, positivity was predominantly epithelial without restriction of staining to certain tissue types, as fittingly indicated by our RT-PCR analysis. Staining was not limited to the cytoplasm but also included nuclear sites. To examine the suitability of the labeled lectin as a histochemical probe we biotinylated galectin-2 under activity-preserving conditions and introduced it to tissue profiling. Specific cytoplasmic staining proved the validity of the concept. Our results encourage systematic histopathologic studies by immuno- and lectin histochemistry, especially by adding galectin-2 as study object to galectin fingerprinting which has already yielded prognostic information on galectins-1, -3, -4 and -8 and hereby contributed to define functional overlap/divergence in this lectin family.

Antibodies↗

PubMed2003

Mutational analysis of the critical bases involved in activation of the AreR-regulated sigma54-dependent promoter in Acinetobacter sp. strain ADP1.

The areR gene in Acinetobacter sp. strain ADP1 regulates the expression of the areCBA genes, which determine growth on benzyl alkanoates. AreR is a member of the NtrC/XylR family of regulatory proteins as determined by sequence homology. Seventy-nine bases upstream of the start of transcription is a region carrying two overlapping inverted repeat (IR) sequences that we predict to be the AreR binding site, also known as the upstream activator site (UAS). IR1 is a near-perfect (16 of 17 bp) repeat separated by 1 bp, and IR2 consists of 9- and 7-bp perfect repeats with a 3-bp gap, with the central bases of the two arms of the repeat separated by 44 and 22 bp. We report here a method for site-directed mutagenesis of chromosomal genes in ADP1 in which linear fragments generated by overlap extension PCR are used to transform ADP1 via its natural transformation system and recombinants are selected by a marker exchange-eviction strategy with a newly created sacB-Km cassette. This method was used to generate 38 strains with designed mutations in the putative UAS upstream of areCBA. The effects of the mutations on areCBA expression were measured by enzyme assays of benzyl alcohol dehydrogenase (AreB) and by reporter gene assays of lacZ inserted into areA. Substitutions or deletions in IR1 had more deleterious effects upon expression when they were in its central region, which overlaps the left arm of IR2, than when they were in its outer regions. By contrast, substitutions in the right arm of IR2 resulted in mutants with relatively high expression levels compared to that of the wild type. Effects of deletions in the right arm of IR2 were very dependent upon the length of the deletion, with 3- or 5-bp deletions reducing expression by >90% whereas an 11-bp deletion in the same area reduced the expression levels by only 50%, suggesting that alterations in the distance and the orientation of the UAS relative to the -24, -12 sigma(54) promoter are critical.

Actinobacteria↗

PubMed2006

Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme.

Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZalphaC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.

Amino Acid Sequence↗

PubMed1994

Polymerase chain reaction (PCR) as a diagnostic tool in HIV infection.

We set up a PCR laboratory for the diagnosis of HIV-1. Probably due to the variability of the HIV-genome, classical primers that performed well in some laboratories in the past, did not suffice for detection of HIV-1 strains in Belgian hospitals. Two new primer sets amplifying a fragment in the LTR-gag gene and in the env gene, which perform better on strains seen in Belgium, have been developed and evaluated. One primer set, conceived and evaluated on Belgian strains by the "Instituut voor Tropische Geneeskunde" in Antwerp, was also included. These three primer sets performed superior (92% sensitivity and 100% specificity on 24 samples) than the classical primers (83.5% sensitivity and 56% specificity on 21 samples). Together with a well-studied testing algorithm, they allow the reliable identification of the presence of the HIV-1 genome. To detect resistance of HIV-1 to reverse transcriptase (RT) inhibitors, we developed a set of two overlapping nested PCR primer sets and additional sequencing primers to amplify and sequence the total RNA or DNA RT gene using a direct cycle sequencing approach of the amplified fragment. Some clinical isolates were amplified and sequenced. In HIV-1 isolates from TIBO R82913-treated patients we identified two amino acid mutations (V108I and Y188L) involved in resistance (more than 100-fold reduced sensitivity). In an untreated patient we identified an amino acid variant (I/V 179D) involved in a 7-fold reduced sensitivity to TIBO. Several other amino acid variants, not involved in resistance, were detected in treated and untreated patients. Using this sequencing technique on cultured virus isolates we also observed in one TIBO-treated patient a differential selection among the strains of the original HIV-1 pool. From this patient we isolated and sequenced a completely TIBO sensitive HIV-1 strain after extensive cultivation in cord blood lymphocytes of the original TIBO resistant HIV-1 virus pool. We could however identify the resistant genotype after cultivation of this resistant HIV-1 virus pool on CEM cells. Our study revealed that sequencing investigations on emerging resistance should preferentially be done with uncultured patient samples since viral sequences and virus-drug sensitivities obtained from isolates cultured in vitro may not necessarily correspond to the sequences and sensitivities of the dominant strain in vivo.

Amino Acid Sequence↗

PubMed1997

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors.

There are already reports, from clinical trials with human immunodeficiency virus type 1 protease inhibitors, of the emergence of drug-resistant mutants which have one or more point mutations in their protease genes. To examine roles of individual and multiple amino acid substitutions in terms of altered enzyme and virus drug sensitivities, we have produced matched vectors for bacterial expression and virus production. Both vectors accept the same restriction enzyme fragment, produced by PCR or PCR-mutagenesis of the protease gene, allowing parallel expression of mutant enzymes in Escherichia coli and in recombinant viruses. The utility of this vector system was demonstrated by using protease variants glycine to valine at amino acid 48 (G48V) and leucine to methionine at amino acid 90 (L90M) identified after passage of HIV-1 in the Roche phase II clinical trial protease inhibitor Ro 31-8959 (H. Jacobsen, K. Yasargil, D. L. Winslow, J. C. Craig, A. Krohn, I. B. Duncan, and J. Mous, Virology 206:527, 1995). G48V, L90M, and G48V/L90M exhibited successively less processing in vitro than the wild-type enzyme, and the purified enzymes were 220-, 20-, and 720-fold, respectively, less sensitive to Ro 31-8959. The reduced enzyme sensitivity correlated directly with the sensitivities of the matched recombinant viruses, in that individual mutations L90M and G48V conferred 2-fold and 4- to 6-fold increases in 50% inhibitory concentration, respectively, whereas G48V/L90M was 8 to 10 times less sensitive to Ro 31-8959. A proviral vector with the entire protease gene deleted was constructed for use as an in vivo recombination target for an overlapping protease PCR fragment, generating wild-type infectious virus. Finally, direct ligation of restriction fragments, generated from random PCR mutagenesis, into the proviral vector should provide a library of protease mutations that allow extremely rapid selection of highly resistant viral variants.

Amino Acid Sequence↗

PubMed1996

The sequence of 23 kb surrounding the SNF3 locus on the left arm of yeast chromosome IV reveals the location of five known genes and characterizes at least six new open reading frames including putative genes for ribosomal protein L35 and a sugar transport protein.

The nucleotide sequence of 22,846 bp of the left arm of chromosome IV is described. Twelve open reading frames (ORFs) greater than 100 triplets were detected, one of which extends into an adjacent cosmid. Two of the ORFs may contain an intron. One of these is an L35 ribosomal protein gene. Five ORFs (D1204, D1214, D1219, D1234 and D1244) encode previously sequenced genes (MGT1, SHM1, ASF2, SNF3 and ARF2, respectively). The nucleotide sequence of a sixth ORF (D1229) is quite similar to the WEB1 gene, which appeared in the DNA databases shortly after finishing the sequence reported here. It is not clear whether or not WEB1 and D1229 represent one and the same gene. The co-linearity of the reported DNA sequences with the genome of strains from Saccharomyces cerevisiae subspecies carlsbergensis, sake and diastaticus was assessed by comparative PCR with overlapping primer sets.

Chromosome Mapping↗