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At least 109 records · Page 6Linked to original sources
PubMed1995

Adenovirus vectors as recombinant viral vaccines.

Adenoviruses can efficiently induce immunity in the lung following single enteric delivery. These viruses can also be engineered to express a number of heterologous proteins in vitro. In the past 10 years, recombinant adenoviruses expressing a variety of antigens have been constructed and tested. This article reviews the main properties of adenoviruses which render them attractive for vaccine development, as well as the results of the immunization studies performed to date. Some disadvantages of this technology and the desired characteristics of second generation adenoviral vectors are discussed.

Adenoviridae↗
PubMed1997

Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes.

Human keratinocyte immortality is genetically recessive to the normal phenotype of limited replicative lifespan and appears to require the dysfunction of p53 and the cyclin D-Cdk inhibitor p16. In order to test for the inactivation of other candidate replicative lifespan genes in the immortal cells of human tumors, we developed a series of mortal and immortal keratinocyte cultures derived from neoplastic lesions of the head and neck which were amenable to molecular genetic analysis by the loss of heterozygosity (LOH) technique. The results indicate that keratinocyte immortalization in head and neck squamous cell carcinoma (SCC-HN) development involves the inactivation of at least two further pathways to senescence and four in all. Chromosomes 1, 4 and 7 carry genes representing immortality complementation groups C, B and D respectively and immortal keratinocytes showed LOH at either 4q32-q34 between D4S1554 and D4S171 (group B) or 7q31 (group D) but never 1q25 (group C). These results tentatively suggest that the genes responsible for the immortality complementation groups encode proteins on the same pathway to senescence. In addition, all of the immortal keratinocyte lines possessed high levels of telomerase activity and a suppressor of telomerase activity has been mapped to the short arm of chromosome 3p. Five out of eight lines showed LOH at 3p21.2-p21.3, a region which may carry a gene capable of suppressing SCC-HN telomerase. However, alternative mechanisms of telomerase reactivation were also suggested by our results. None of the above genetic alterations were seen in seven senescent neoplastic keratinocyte cultures. Other loci harbouring antiproliferative genes implicated in replicative lifespan showed few or no alterations and any alterations seen were additional to those described above.

Carcinoma, Squamous Cell↗
PubMed2005

Characterization and molecular mapping of genes determining semidwarfism in barley.

The semidwarf trait is desired in cereal breeding programs for increased lodging resistance. We characterized 27 brachytic (brh) semidwarf mutants in barley (Hordeum vulgare L.) and located the genes on barley chromosome linkage maps. All brachytic genes were transferred into the two-rowed cultivar Bowman by backcrossing four to seven times and selecting for semidwarf plants. The brachytic lines were evaluated for 10 phenotypic traits: plant height, awn, peduncle, and rachis internode length, leaf length and width, lodging, grain yield, number of kernels per spike, and kernel weight. We intercrossed the lines to determine which mutants were at independent loci and which were alleles at the same locus. F2 populations from 18 brh semidwarfs were constructed for genetic mapping using simple sequence repeat (SSR) markers. The brachytic semidwarf near-isogenic lines were significantly shorter than their normal counterparts and most had lower yields (16/27); shorter awns (26/27), peduncles (26/27), and rachis internodes (24/27); and reduced kernel weight (22/27). Twelve of the lines had shorter penultimate leaves and 15 had reduced lodging. Four lines had increased kernels per spike, while one had fewer kernels per spike. Allelism tests and mapping comparisons indicated that the 27 semidwarfs comprise 18 independent genetic loci. SSR mapping placed these loci in five of the seven barley chromosomes. Knowledge of the effects and locations of these brachytic semidwarf genes will help barley breeders select appropriate lines for barley improvement.

Chromosome Mapping↗
PubMed1994

Genetic requirements for initiating asexual development in Aspergillus nidulans.

Conidiation in the filamentous ascomycete Aspergillus nidulans requires activation of brlA, a well-characterized transcriptional regulator of genes that are induced specifically during asexual development. We have isolated and characterized developmental mutations in six loci, designated fluG, flbA, flbB, flbC, flbD, and flbE, that result in defective development and reduced brlA expression. These mutants grow indeterminately to produce masses of aerial hyphae resulting in the formation of cotton-like colonies with a "fluffy" morphology. The results of growth and epistasis tests involving all pairwise combinations of fluffy mutations indicate complex hierarchical relationships among these loci. We discuss these genetic interactions and propose that there are multiple mechanisms for activating brlA.

Alleles↗
PubMed1986

Enhanced synthesis of the glucose/calcium-regulated proteins in a hamster cell mutant deficient in transfer of oligosaccharide core to polypeptides.

The properties of two Chinese hamster temperature-sensitive mutants, K12 and H3.5, were examined. Both mutants originated from the same parental cell line, Wg1A, and were isolated as cell cycle mutants arrested in G1. Previously, we had been shown that the H3.5 ts mutation affected the transfer of the oligosaccharide from the lipid carrier to the nascent polypeptide and that the K12 ts mutation regulated the transcription of two glucose/calcium-regulated genes. We report here that these two mutants exhibit almost identical phenotypes at the biochemical level. Furthermore, a genetic complementation test demonstrates that the two ts lesions must be closely related, or even identical. Our results suggest that a specific defect in glycosylation may result in the overproduction of the glucose/calcium-regulated proteins and is capable of activating the promoter of the major glucose-regulated gene.

Animals↗
PubMed1985

Fuzzy: a genetic marker for warfarin resistance in the Norway rat.

No recombination was observed between fuzzy (fz) and warfarin resistance (Rww) in 245 rats from a backcross test for linkage. This locates fz very close to Rw in linkage group I. The map position established for fz makes this allele particularly suitable for use in combination with p (pink-eyed dilution) for linkage tests in group I and as a genetic marker for the Rw locus in studies of warfarin resistance. The map position of fz in the rat corresponds to that of fr (frizzy) in the mouse, suggesting homology between these loci in the 2 species.

Animals↗
PubMed1995

Recombinant retroviral vector interferes with the detection of amphotropic replication competent retrovirus in standard culture assays.

Many protocols for gene therapy employ recombinant retroviral vectors, which are replication-defective retroviruses engineered to serve as gene delivery vehicles. The use of retroviral vectors for human gene therapy requires careful screening of vector-producing cell lines and culture supernatants to ensure the absence of replication competent retrovirus (RCR) in clinical products. In this study we have examined several different culture assays routinely used to test for the presence of RCR. Results indicate that cocultivation of a vector-producing cell line with a permissive cell line can reproducibly detect a low level of contaminating RCR. RCR was detected less frequently in direct tests of cell-free culture supernatants from a contaminated vector-producing line. Further studies revealed that recombinant retroviral vector can interfere, to varying degrees, with the detection of low-level RCR in culture supernatants when a marker rescue assay, an extended mink S+L- assay or a PG-4 S+L- assay is used. Interference can be partially overcome by culturing the vector preparation with a permissive cell line for several days before testing on the indicator cell line. The interference phenomenon we have observed may also occur in other culture assays routinely used for the detection of RCR.

Animals↗
PubMed1983

High proportion of multi-locus deletions among hycanthone-induced X-linked recessive lethals in Drosophila melanogaster.

328 X-linked recessive lethal mutations induced in late spermatids by hycanthone methanesulfonate were tested for coverage by duplications that comprised, in total, about 24% of the euchromatic X chromosome; 78 lethals appeared to be covered. Crossover localization tests of a random sample of 38 non-covered lethals revealed 4 chromosomes carrying a lethal within a duplicated segment. Lethals localized to a particular region were crossed to reference deficiencies and single-locus mutations, and inter se, to ascertain their genetic extent. The proportion of multi-locus deletions among these 78 covered and 4 non-covered lethals was 3/48, 1/10 and 13/24 for the distal, medial and proximal regions, respectively. A storage period of 9 days did not noticeably influence these proportions. In the sample of 38 non-covered lethals, and among 17 of the covered single-site lethals, 4 cases of strong crossover suppression were detected. Comparison of these results with data obtained with other mutagens suggests that induction of multi-locus deletions, and possibly of other types of chromosome rearrangement, could in part depend on other mechanisms than those acting in the formation of translocations and chromosome loss. For the purpose of mutagen testing, these findings imply that, in Drosophila, results in the regular genetic tests for chromosome breakage events do not always accurately predict the capacity of a mutagen to induce multi-locus deletions. This is of importance since transmissible multi-locus deletions have been considered a significant source of genetic damage in man.

Animals↗
PubMed1995

Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010.

The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.

Agrobacterium tumefaciens↗
PubMed1992

Co-recessive inheritance: a model for DNA repair and other surveillance genes in higher eukaryotes.

The co-recessive inheritance hypothesis proposes that certain recessively inherited diseases require homozygosity and/or hemizygosity for defective alleles at more than one locus simultaneously for the trait to be expressed. Although this hypothesis was originally proposed in the context of defective alleles for genes coding for DNA-repair functions, it need not be limited to this context, and genetic selection pressure may favor this model for genes involved in surveillance of any type. The co-recessive inheritance hypothesis also predicts extremely high carrier frequencies, likely affecting much of the general population, for defective alleles associated with these rare recessive diseases. The model predicts much lower rates of consanguinity between the parents of affected individuals than autosomal recessive inheritance, allowing it to be tested epidemiologically, and recent data suggest that the hypothesis may be valid for some cases of ataxia telangiectasia and xeroderma pigmentosum. The model provides possible explanations for a number of otherwise puzzling findings in several diseases associated with defective DNA repair.

Animals↗
PubMed1992

Cytogenetic analysis of chromosome region 89A of Drosophila melanogaster: isolation of deficiencies and mapping of Po, Aldox-1 and transposon insertions.

We have initiated a cytogenetic analysis of chromosome region 89A of Drosophila melanogaster by isolating a set of radiation-induced mutations causing loss of function of P[(w)B]1-1, a transposon bearing the white locus inserted in 89A. Complementation tests and cytological examination of these chromosomes identified four new deficiencies (Df(3R)Po2, Df(3R)Po3, Df(3R)Po4 and Df(3R)c(3)G2). The new deficiencies and three previously identified deficiencies (Df(3R)sbd26, Df(3R)sbd45 and Df(3R)sbd105) were tested for the ability to complement mutations in the enzyme loci Po and Aldox-1, the indirect flight muscle genes Tm2 and act88F, the morphological mutations jvl, sbd2 and Sb, the vital loci srp, pnr and mor, and a newly described vital locus l(3)89Aa. We also used linkage analysis to determine the order and relative positions of P[(w)B]1-1 and an independent transposon insertion, P[w+]21, with respect to cv-c, Po, Aldox-1 and sbd2. Cytological examination of the deficiencies and analysis of the transformed lines by in situ hybridization permits the correlation of genetically defined regions with specific polytene chromosome bands. A revised cytogenetic map of the 88F-89B region is presented.

Aldehyde Oxidase↗
PubMed1994

An "instant gene bank" method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA.

We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis. This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple. The method relies on recombination between a fungal replicating vector pHELP1 and linear G. graminis genomic DNA during co-transformation. We were able to complement two out of seven A. nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli. Complementation of the A. nidulans argB mutation resulted from integration of 8-10 kb segments of G. graminis DNA into pHELP1. The complementation of the A. nidulans pyrG mutation resulted from a complex rearrangement. Complementing DNA was shown to originate from G. graminis, and was capable of retransforming the original mutants to give the expected phenotype.

Aspergillus nidulans↗
PubMed1977

Characterization of glycerol nonutilizing and protoperithecial mutants of Neurospora.

Mutants defective in polyol metabolism and/or in protoperithecial development were selected in Neurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed into N. crassa to facilitate genetic analysis. One, glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, between ad-1 and rib-1; the other, glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal to ad-9. Another mutant, gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal to ad-9.

Chromosome Mapping↗
PubMed2001

Molecular and functional mapping of the piebald deletion complex on mouse chromosome 14.

The piebald deletion complex is a set of overlapping chromosomal deficiencies surrounding the endothelin receptor B locus collected during the Oak Ridge specific-locus-test mutagenesis screen. These chromosomal deletions represent an important resource for genetic studies to dissect the functional content of a genomic region, and several developmental defects have been associated with mice homozygous for distinct piebald deletion alleles. We have used molecular markers to order the breakpoints for 20 deletion alleles that span a 15.7-18-cM region of distal mouse chromosome 14. Large deletions covering as much as 11 cM have been identified that will be useful for regionally directed mutagenesis screens to reveal recessive mutations that disrupt development. Deletions identified as having breakpoints positioned within previously described critical regions have been used in complementation studies to further define the functional intervals associated with the developmental defects. This has focused our efforts to isolate genes required for newborn respiration and survival, skeletal patterning and morphogenesis, and central nervous system development.

Alleles↗
PubMed2003

Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus.

LacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of approximately 13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside).

Base Sequence↗
PubMed1992

Mutations affecting sensitivity of the cellular slime mold Dictyostelium discoideum to DNA-damaging agents.

We describe 22 new mutants of D. discoideum that are sensitive to DNA damage. These mutants were isolated on the basis of sensitivity to either temperature, gamma-rays, or 4-nitroquinolone-1-oxide (4NQO). The doses of gamma-rays, ultraviolet light (UV), and 4NQO required to reduce the survival of colony-forming ability of these mutants to 10% (D10) range from 2% to 100% of the D10s for the nonmutant, parent strains. For most of the mutants, those which are very sensitive to one agent are very sensitive to all agents tested and those which are moderately sensitive to one agent, are moderately sensitive to all agents tested. One mutant is sensitive only to 4NQO. Linkage relationships have been examined for 13 of these mutants. This linkage information was used to design complementation tests to determine allelism with previously characterized complementation groups affecting sensitivity to radiation. 4 of the new mutants fall within previously identified complementation groups and 3 new complementation groups have been identified (radJ, radK and radL). Other new loci probably also exist among these new mutants. This brings the number of characterized mutants of D. discoideum which are sensitive to DNA-damaging agents to 33 and the number of assigned complementation groups to 11.

4-Nitroquinoline-1-oxide↗
PubMed1978

The role of X-linked lethal and viable male-sterile mutations in male gametogenesis of Drosophila melanogaster: genetic analysis.

The possibility that viable male-sterile mutations occur in vital genes and the role played by lethal mutations and viable male-steriles in male gametogenesis were studied. Five sterile loci were identified among the 30 most proximal vital loci of the X-chromosome and two of them were shown to be allelic with lethal mutations. Fertility test on gynanders for nonautonomous lethal mutations proved that vital genes operate autonomously in male gonads, independently of their effect on somatic tissues. Fertility tests of ts lethals, shifted to the nonpermissive temperature after the TSP, showed that 40% of vital genes function in male gonads. It is further shown that about the same proportion of vital genes is operating in female gonads and that the two groups overlap by about 70%. The role of viable and lethal male gametogenesis is discussed in detail.

Drosophila melanogaster↗
PubMed2000

Functional and genomic analysis of the human mitochondrial intermediate peptidase, a putative protein partner of frataxin.

We showed recently that the yeast mitochondrial intermediate peptidase (YMIP polypeptide; gene symbol, OCT1) promotes mitochondrial iron uptake by catalyzing the maturation of iron-utilizing proteins and exacerbates the mitochondrial iron accumulation that results from loss of yeast frataxin, a mitochondrial protein required for mitochondrial iron efflux. This suggests that the human MIP (HMIP polypeptide; gene symbol MIPEP) may be one of the loci predicted to influence the clinical manifestations of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease caused by lack of human frataxin. To begin to test this hypothesis, we have characterized HMIP at the functional and genomic levels. We show that HMIP can complement a yeast knock-out mutant lacking YMIP, demonstrating that HMIP and YMIP are functional homologues. The MIPEP gene spans 57 kb and consists of 19 exons that correlate with the functional domains of HMIP. Primer extension analysis has identified a major transcript of the MIPEP gene expressed differentially and predominantly in tissues with high oxygen consumption, while sequence analysis of approximately 2 kb of 5'-flanking DNA has revealed putative Mt1/3/4, NF-kappaB, and AP-1 elements that may regulate MIPEP expression in these tissues. Using a new polymorphic (CA)(n) repeat in intron 4, MIPEP has been genetically mapped within a 7-cM interval between markers D13S283 and D13S217 on 13q12. This work provides the basis for molecular analysis of MIPEP in FRDA and possibly other neurodegenerative diseases.

Base Sequence↗