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PubMed1998

An enhanced packaging system for helper-dependent herpes simplex virus vectors.

Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types. The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (pac) elements have been deleted. Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles. Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector. The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in Escherichia coli from which the pac elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome. The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector. The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination. The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield.

Animals↗
PubMed2001

Identification of the rhesus macaque rhadinovirus lytic origin of DNA replication.

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.

Animals↗
PubMed2001

African swine fever virus multigene family 360 and 530 genes are novel macrophage host range determinants.

Pathogenic African swine fever virus (ASFV) isolates primarily target cells of the mononuclear-phagocytic system in infected swine and replicate efficiently in primary macrophage cell cultures in vitro. ASFVs can, however, be adapted to grow in monkey cell lines. Characterization of two cell culture-adapted viruses, MS16 and BA71V, revealed that neither virus replicated in macrophage cell cultures. Cell viability experiments and ultrastructural analysis showed that infection with these viruses resulted in early macrophage cell death, which occurred prior to viral progeny production. Genomic cosmid clones from pathogenic ASFV isolate E70 were used in marker rescue experiments to identify sequences capable of restoring MS16 and BA71V growth in macrophage cell cultures. A cosmid clone representing a 38-kbp region at the left terminus of the genome completely restored the growth of both viruses. In subsequent fine-mapping experiments, an 11-kbp subclone from this region was sufficient for complete rescue of BA71V growth. Sequence analysis indicated that both MS16 and BA71V had significant deletions in the region containing members of multigene family 360 (MGF 360) and MGF530. Deletion of this same region from highly pathogenic ASFV isolate Pr4 significantly reduced viral growth in macrophage cell cultures. These findings indicate that ASFV MGF360 and MGF530 genes perform an essential macrophage host range function(s) that involves promotion of infected-cell survival.

African Swine Fever Virus↗
PubMed2002

The herpes simplex virus type 1 US11 protein binds the coterminal UL12, UL13, and UL14 RNAs and regulates UL13 expression in vivo.

The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. US11 localizes to the nucleolus in infected cells, can associate with ribosomes, and has been shown to bind RNA. The RNA substrates of US11 identified thus far have no apparent role in the virus lytic cycle, so we set out to identify a novel, biologically relevant RNA substrate(s) for this protein in HSV-1-infected cells. We designed a reverse transcriptase PCR-based protocol that allowed specific selection of a 600-bp RNA binding partner for US11. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14. We show that the binding of US11 to 12/14 is sequence-specific and mediated by the C-terminal domain of the protein. To elucidate the role of US11 in the virus life cycle, we infected cells with wild-type virus, a cosmid-reconstructed US11 HSV-1 null mutant, and a cosmid-reconstructed wild-type virus and analyzed expression of UL12, -13, and -14 during a time course of infection. These experiments revealed that this interaction has biological activity; at early times of infection, US11 down-regulates UL13 protein kinase mRNA and protein.

Base Sequence↗
PubMed2002

Novel swine virulence determinant in the left variable region of the African swine fever virus genome.

Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70DeltaNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70DeltaNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDeltaNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalDeltaNL genome was capable of restoring full virulence to E70DeltaNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DeltaNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalDeltaNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70DeltaNL. Comparative nucleotide sequence analysis of the left variable region of the E70DeltaNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70DeltaNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalDeltaNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.

African Swine Fever↗
PubMed2004

Expansion of a unique region in the Marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation.

Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Delta132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Delta132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Delta132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.

Animals↗
PubMed1984

Conversion through homologous recombination of the gene encoding Simian virus 40 115,000-molecular-weight super T antigen to a gene encoding a normal-size large T antigen variant.

We have previously cloned the gene encoding a 115,000-Mr super T antigen (115K super T antigen), an elongated form of the Simian virus 40 large T antigen, originating from the rat cell line V 11 F1 clone 1, subclone 7 (May et al., J. Virol. 45:901-913, 1983). DNA sequence analysis has shown that the 115K super T antigen gene contains notably an in-phase duplication of a sequence located in the region of tsA mutations. We have also shown that the 115K super T antigen gene is able to induce the formation of transformed foci in transfected rat cells. After rat cell cultures were transfected with the cloned gene encoding 115K super T antigen, we obtained a large number of transformants as reported in this paper. In these transformants, we detected a very high frequency of new T antigen variants, as shown by immunoprecipitation of the cell extracts with anti-simian virus 40 tumor serum followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Based on these results and all of the data presently available, it appears likely that the input plasmid or cosmid DNAs containing the cloned gene were first subjected to recombination events that yield new variant T antigen genes before these recombinant genes become integrated. The new variant T antigens observed in the transformants were predominantly those comigrating with normal-size large T antigen. In fact, these latter variants appeared to be indistinguishable from wild-type large T antigen as judged by restriction mapping by Southern blotting of the total genomic DNA of the transformants. Models of intermolecular or intramolecular homologous recombination occurring between or within the input plasmid or input cosmid DNA molecules are proposed to account for the formation of such revertants.

Animals↗
PubMed1985

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

Alleles↗
PubMed1986

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.

Cell Cycle↗
PubMed1988

The brown protein of Drosophila melanogaster is similar to the white protein and to components of active transport complexes.

The brown gene of Drosophila melanogaster is required for deposition of pteridine pigments in the compound eye and other tissues. We isolated a ca. 150-kilobase region including brown by microdissection and chromosome walking using cosmids. Among the cDNAs identified by hybridization to the cosmids, one class hybridized to a genomic region that is interrupted in two brown mutants, bw and In(2LR)CK, and to 2.8- and 3.0-kilobase poly(A)+ RNAs which are altered in the mutants. Nucleotide sequencing of these cDNAs revealed that the two transcripts differ as a consequence of alternative poly(A) addition and that both encode the same predicted protein of 675 amino acids. Searches of available databases for amino acid sequence similarities detected a striking overall similarity of this predicted protein to that of the D. melanogaster white gene. The N-terminal portion aligned with the HisP family of membrane-associated ATP-binding proteins, most of which are subunits of active transport complexes in bacteria, and to two regions of the multidrug resistance P-glycoprotein. The C-terminal portion showed a structural similarity to integral membrane components of the same complexes. Taken together with earlier biochemical evidence that brown and white gene products are necessary for uptake of a pteridine precursor and genetic evidence that brown and white proteins interact, our results are consistent with suggestions that these proteins are subunits of a pteridine precursor permease.

ATP-Binding Cassette Transporters↗
PubMed1996

FISH studies in a patient with sporadic aniridia and t(7;11) (q31.2;p13).

A 2 year old female presenting with bilateral sporadic aniridia was found to have an apparently balanced reciprocal translocation with a chromosome 11 breakpoint within band p13. Fluorescence in situ hybridisation (FISH) studies with distal 11p13 specific cosmids showed that the chromosome 11 breakpoint lay between the aniridia (PAX6) locus and a region approximately 100 kb distal to PAX6 defined by the cosmid FO2121. Although this patient did not have a detectable deletion within PAX6, her aniridia may have resulted from a disruption of the distal chromatin domain containing either enhancers or regulators for PAX6. This case may therefore be another example of aniridia caused by a position effect as recently described in two familial aniridia patients in which the phenotype cosegregated with chromosome abnormalities with 11p13 breakpoints.

Aniridia↗
PubMed1998

Severe phenotype resulting from an active ring X chromosome in a female with a complex karyotype: characterisation and replication study.

We report on the characterisation of a complex chromosome rearrangement, 46,X,del(Xq)/47,X,del(Xq),+r(X), in a female newborn with multiple malformations. Cytogenetic and molecular methods showed that the del(Xq) contains the XIST locus and is non-randomly inactivated in all metaphases. The tiny r(X) chromosome gave a positive FISH signal with UBE1, ZXDA, and MSN cosmid probes, but not with a XIST cosmid probe. Moreover, it has an active status, as shown by a very short (three hour) terminal BrdU pulse followed by fluorescent anti-BrdU antibody staining. The normal X is of paternal origin and both rearranged chromosomes originate from the same maternal chromosome. We suggest that both abnormal chromosomes result from the three point breakage of a maternal isodicentric idic(X)(q21.1). Finally, the phenotype of our patient is compared to other published cases and, despite the absence of any 45,X clone, it appears very similar to those with a 45,X/46,X,r(X) karyotype where the tiny r(X) is active.

Abnormalities, Multiple↗
PubMed1991

Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa.

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential alpha-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.

Amino Acid Sequence↗
PubMed1992

Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity.

Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12. Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P. cepacia. Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin. In addition, this mutant lost its ability to antagonize fungal phytopathogens. Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P. cepacia. However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E. coli to P. cepacia.

Antifungal Agents↗
PubMed1993

Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs.

We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifluorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and, to the human eye, the green signals appeared less bright. With a blue fluorescent coumarin-labeled dUTP, only highly repetitive target sequences could be visualized directly. Imaging with a cooled CCD (charged coupled device) camera for prolonged integration times permitted localization of small targets using probes labeled with red and green fluorochromes. Visualization of a 1.8-kb single copy sequence with a rhodamine-labeled cDNA proved feasible. The strategy of identifying hybridization sites with multiple colors was successfully applied for multiple hybridizations to repetitive and unique targets, using the red and green labels directly and the biotin label indirectly with one layer of avidin-coumarin.

Cells, Cultured↗
PubMed1995

Mapping of the gene for hormone sensitive lipase (LIPE) to chromosome 19q13.1-->q13.2.

Free fatty acids are the major source of fuel for mammals, and hormone sensitive lipase (LIPE) plays a critical role in lipid metabolism by mobilizing free fatty acids from stored triglycerides. We have identified and sequenced a partial cDNA for LIPE. Cosmids were identified by hybridization and mapped to 19q13.1-->q13.2 by FISH. Direct sequence analysis of a 1 kb segment of cosmid 26710 identifies a dinucleotide repeat in an intron upstream of exon 8 of human LIPE. This marker was heterozygous 82% of the time with 12 alleles (166-190 bp) detected in 122 chromosomes. The most likely order for this gene is: qter-[D19S178/LIPE]-(3 cM)-D19S47-(1 cM)-D19S190-RYR1-cen.

Alleles↗
PubMed1996

SOX20, a new member of the SOX gene family, is located on chromosome 17p13.

SOX genes share a high sequence identity with the HMG box present in the testis determining gene SRY. We have identified a HMG box-like sequence motif on six contiguous cosmids, which cross-hybridize to a SOX9 cDNA probe. A data base search revealed a high similarity of the deduced amino acid sequence to the human SOX12 and the murine Sox16 HMG domains. The cosmids were assigned to chromosome 17p13 by FISH analysis.

Amino Acid Sequence↗
PubMedDate not supplied

Fluorescence in situ hybridization (FISH) as a method to detect aneuploid cells.

OBJECTIVES: To test the sensitivity and specificity of various FISH probes for detecting male and aneuploid cells and to determine the percentage of fetal cells that must be present in a sample in order to use the probes for prenatal diagnosis. METHODS: Adult human lymphocytes were cultured and harvested. Twelve different proportions of male to female cells and 5 different proportions of trisomy 21 cells and trisomy 18 cells in euploid cells were prepared for FISH. Alpha-satellite DYZ1 was applied to detect the male cells. Chromosome 21/Down syndrome critical region cosmid and D13Z1/D21Z1 alpha-satellite probes were applied to detect trisomy 21 cells. The D18Z1 alpha-satellite probe was used to detect trisomy 18 cells. RESULTS: DYZ1 detected male cells reliably in concentrations as low as 2%. Both D18Z1 and chromosome 21/Down syndrome critical region cosmid probes could detect aneuploid cells at reasonably achievable concentrations. However, the D13Z1/D21Z1 probe was not sensitive below concentrations of 50%. CONCLUSIONS: FISH is an alternative technique for noninvasive prenatal diagnosis. The sensitivity and specificity of FISH probes may play a crucial role in the accuracy of prenatal diagnosis.

Adult↗

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