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PubMed1993

Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae.

The chromatin structure of yeast ribosomal DNA was analyzed in vivo by crosslinking intact cells with psoralen. We found that in exponentially growing cultures the regions coding for the 35S rRNA precursor fall into two distinct classes. One class was highly accessible to psoralen and associated with nascent RNAs, characteristic for transcriptionally active rRNA genes devoid of nucleosomes, whereas the other class showed a crosslinking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rRNA gene copies. By crosslinking the same strain growing in complex or minimal medium, we have shown that yeast cells can modulate the proportion of active (non-nucleosomal) and inactive (nucleosomal) rRNA gene copies in response to variations in environmental conditions which suggests that yeast can regulate rRNA synthesis by varying the number of active gene copies, in contrast to the vertebrate cells studied so far. Whereas intergenic spacers flanking inactive rRNA gene copies are packaged in a regular nucleosomal array, spacers flanking active genes show an unusual crosslinking pattern suggesting a complex interaction of regulatory factors and histones with DNA.

Chromatin↗
PubMed2002

Clonal proteomics: one gene - family of proteins.

The work presented here attempts to consolidate our knowledge on cellular transcriptome and proteome. It takes into account that a typical activated cell (lymphocyte) contains 40 000 mRNA molecules at any time, and it represents about 5000 different molecular species of transcripts. Such a cell has about 1 000 000 000 protein molecules, some of them being present at 10 000 000 copies while others at a very low copy number (say 1 to 10 copies per cell). By studying cell free expression of individual cDNA clones (or pools of known complexity) we address to those rare molecular components that will remain undetected by the current analytical means. For our analysis we use cell free translation systems (wheat germ or rabbit reticulocyte origin) and we study polypeptide products originating from intact, or restriction endonuclease-treated cDNA clones. We conclude that in most instances expressed genes yield transcript(s) that translate into several, and often very numerous families of polypeptide species. In our ISODALT two-dimensional gel system we characterize the proteomic profile of the clonal polypeptide families in terms of their molecular mass, charge, multiple products, and appearance.

Animals↗
PubMed1999

Mechanism of action of the Rep protein from the Dictyostelium Ddp2 plasmid family.

The two-hybrid system was used to show that the Rep proteins from three members of the Dictyostelium discoideum Ddp2 plasmid family, Ddp2, Ddp5, and Ddp6, form homomultimers but not heteromultimers when expressed in yeast cells. The results with deletion mutations suggest that multiple regions of the Rep proteins are involved in the multimerization. Electrophoretic mobility shift assays with heterologously expressed and purified Ddp2 Rep protein showed that it is a DNA binding protein. The nucleosomal organization of Ddp2 and Ddp6 in their inverted repeat and promoter regions was investigated. Analysis of mutants derived from the Ddp6 plasmid revealed that its Rep protein is required for nucleosome positioning (i.e., phasing) to occur in the promoter region. On the other hand, nucleosome positioning in the inverted repeat regions of both plasmids is not dependent on Rep protein but on either a feature of the DNA sequence or the binding of cellular factors, perhaps the Dictyostelium origin recognition complex. Rep protein is likely involved in transcription regulation and control of DNA replication, specifically amplification of plasmid at low copy numbers. The formation of homomultimers may be required for their regulatory activity.

Animals↗
PubMed1995

Mechanisms of resistance to combinations of vincristine, etoposide and doxorubicin in Chinese hamster ovary cells.

We have isolated from Chinese hamster ovary cells, 30 sublines resistant to vincristine, doxorubicin or etoposide and 43 sublines evading treatment with a pair of these drugs. Isolated in one step and under low selective pressure, sublines were 3- to 25-fold more resistant to their selecting drug(s) than the parental cells. Possible P-glycoprotein-associated multidrug resistance was investigated through pgp gene copy number and mRNA expression level. DNA topoisomerase II alteration was evaluated from the ability of nuclear extracts to form cleavable complexes. Vincristine (all sublines) and doxorubicin (6/7 sublines) preferentially selected for pgp gene amplification and mRNA overexpression, whereas selection with etoposide resulted in a decrease of cleavable complex formation in 11 out of 13 sublines. A common pgp gene-mediated resistance was found in the 13 doxorubicin plus vincristine-selected sublines, whereas all but one of the 12 etoposide plus vincristine-resistant sublines displayed both pgp mRNA overexpression and decreased ability to form cleavable complexes. Among the 18 doxorubicin plus etoposide selected sublines, five exhibited a decreased ability to form cleavable complexes only, six exhibited pgp mRNA overexpression only and six exhibited both alterations. Overall, drug resistance could not be attributed to either mechanism in three of the 73 sublines. We conclude that under low selective pressure it is possible to find a combination of drugs which require simultaneous selection of more than one resistance mechanism; such cells emerge with very low frequency.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Murine MHC class II locus control region drives expression of human beta-glucocerebrosidase in antigen presenting cells of transgenic mice.

Gaucher disease is the most prevalent lysosomal storage disorder in humans, resulting from an inherited deficiency of the enzyme glucocerebrosidase. Although the enzyme is ubiquitously expressed, cells of the reticuloendothelial system are particularly affected since they accumulate the undigested glucosylceramide substrate through their role in scavenging and breaking down cell debris. Gaucher disease is an attractive target for somatic gene therapy. To test the ability to express the enzyme in the affected cell types we have generated transgenic mice expressing human glucocerebrosidase under the control of the murine major histocompatibility complex (MHC) class II Ead locus control region (LCR). The four transgenic lines express the human enzyme in a copy number-dependent manner, independent of the integration site of the transgene. Over-expression of the human enzyme in mice did not result in any abnormal phenotype or pathology during the period of observation (> 2 years). The enzyme is expressed in B cells, monocytes, dendritic cells, thymic epithelial cells, and macrophages in various tissues: the peritoneal cavity, bone marrow, spleen, kidney, gastrointestinal tract, Kupffer cells in the liver and alveolar macrophages in lungs. Expression in the brain was limited to perivascular macrophages and was not seen in microglial cells. Therefore, the MHC class II LCR could potentially be of use in somatic gene therapy for type 1 Gaucher disease.

Animals↗
PubMed1985

Variable numbers of pepsinogen genes are located in the centromeric region of human chromosome 11 and determine the high-frequency electrophoretic polymorphism.

A panel of 26 mouse-human somatic cell hybrids containing different human chromosome complements was analyzed with a cloned human pepsinogen cDNA probe to determine the chromosomal location and the number of genes encoding these proteins. A complex containing variable numbers of pepsinogen genes was localized to the centromeric region of human chromosome 11 (p11----q13). Examination of somatic cell hybrids containing single copies of chromosome 11 and the corresponding human parental cell lines revealed a restriction fragment length polymorphism determined by pepsinogen haplotypes that contained two or three genes, respectively. Concurrent studies of DNA from individuals exhibiting the most common pepsinogen electrophoretic phenotypes with exon-specific probes demonstrated that the absence of one gene among the different restriction fragment patterns correlated with the absence of one specific isozymogen (Pg 5). Thus, our studies demonstrate that this genetic polymorphism involving intensity variation of individual pepsinogen isozymogens results from chromosome haplotypes that contain different numbers of genes. The regional localization of this polymorphic gene complex will facilitate detailed linkage analysis of human chromosome 11.

Centromere↗
PubMed1998

Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis.

The Mdm2 proto-oncogene is amplified to high copy numbers in human sarcomas and is overexpressed in a wide variety of other human cancers. Because Mdm2 protein forms a complex with the p53 tumor suppressor protein and down-regulates p53 function, the oncogenic potential of Mdm2 is presumed to be p53-dependent. To model these conditions in mice, we have used the entire Mdm2 gene, under transcriptional control of its native promoter region, as a transgene to create mice that overexpress Mdm2. The transgenic mice are predisposed to spontaneous tumor formation, and the incidence of sarcomas observed in the Mdm2-transgenic mice in the presence or absence of functional p53 demonstrates that, in addition to Mdm2-mediated inactivation of p53, there exists a p53-independent role for Mdm2 in tumorigenesis.

Animals↗
PubMed2002

Optimizing anti-CD3 affinity for effective T cell targeting against tumor cells.

Bispecific antibodies binding to the TCR/CD3 complex and to a tumor-associated surface molecule can be used to target cytotoxic T lymphocytes against tumor cells. We reasoned that high-affinity binding to CD3 may reduce the efficiency of T cell stimulation and target the bispecific reagent to T cells rather than to tumor cells in vivo. We therefore mutated a bispecific single-chain antibody (BscAb) directed to human CD3 and EpCAM to generate variants that bind to CD3 with higher or lower affinity. When compared to the wild-type molecule, a mutant with increased binding to CD3 showed lower capacity to target T cells against an EpCAM+ tumour. In contrast, mutants with decreased binding to CD3, in spite of rapid dissociation, efficiently triggered T cell activation and cytotoxicity, especially when present on tumor cells at low copy number. These results are consistent with the TCR serial triggering model and suggest that BscAb with extremely low affinity for the TCR-CD3 complex could be exploited therapeutically because of their preferential localization to tumor cells.

Antibodies, Bispecific↗
PubMed1998

The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA.

Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression of GCN4 mRNA. The phenotypes of gcd10 mutants were suppressed by high-copy-number IMT genes, encoding initiator methionyl tRNA (tRNAiMet), or LHP1, encoding the yeast homolog of the human La autoantigen. The gcd10-504 mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet. Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNAiMet is the essential function of Gcd10p. A gcd14-2 mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMet processing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMet maturation. The chromatographic behavior of elongator and initiator tRNAMet on a RPC-5 column indicated that both species are altered structurally in gcd10Delta cells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable in gcd10Delta tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

Adenosine↗
PubMed1997

Cytogenetic analysis of three breast carcinoma cell lines using reverse chromosome painting.

Chromosome painting was used to determine the copy number and identity of virtually all the chromosomes in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1. The karyotypes of all three cell lines were very complex, and were consistent with the monosomic pattern of evolution suggested by Dutrillaux, in which nonreciprocal translocations cause an initial reduction in chromosome number, followed by duplication of the entire genome and further chromosome loss. Twenty distinct abnormal chromosomes were identified in T-47D, seven of which were present as two copies. MDA-MB-361 had 27 abnormal chromosomes each as a single copy. Thirteen abnormal chromosomes in ZR-75-1 occurred singly, two were paired, and one was present as three copies. Most of the aberrant chromosomes were nonreciprocal translocations, although deletions, duplications, isochromosomes, and amplifications (HSR of 1q) were also found. Chromosome arms present in abnormal chromosomes in all three lines were 1q, 6p, 7p, 8p, 8q, 10q, 11p, 11q, 12p, 13q, 14q, 15q, 16p, 16q, 17q, and 20q. The only chromosome arms present in four or more copies in all three lines were 8q and proximal 12p, while 1p, 17p, and bands 11q12--13 were the only chromosome regions consistently reduced to two copies. The most striking feature common to all three lines was a translocation breakpoint on the short arm of chromosome 8 at 8p12.

Breast Neoplasms↗
PubMed1990

Complex character of senS, a novel gene regulating expression of extracellular-protein genes of Bacillus subtilis.

The senS gene of Bacillus subtilis, which in high copy number stimulates the expression of several extracellular-protein genes, has been cloned, genetically mapped, and sequenced. The gene codes for a highly charged basic protein containing 65 amino acid residues. The gene is characterized by the presence of a transcription terminator (attenuator) located between the promoter and open reading frame, a strong ribosome-binding site, and a strong transcription terminator at the 3' end of this monocistronic gene. The amino acid sequence of SenS showed partial homology with the N-terminal core binding domain region of bacterial RNA polymerase sigma factors and a helix-turn-helix motif found in DNA-binding proteins. The gene can be deleted without any effect on growth or sporulation.

Amino Acid Sequence↗
PubMed1983

Intracellular location of small circular DNA complexes in mammalian cell lines.

For determination of the cellular location of small polydisperse circular DNA complexes, rat myoblastic L6 cells, HeLa cells, and mouse L cells were enucleated and processed by the micapress-adsorption method for electron microscopy (H. Yamagishi, T. Kunisada, and T. Tsuda, 1982, Plasmid 8, 299-306). Small circular DNA complexes from intact cells showed a heterogeneous size distribution of from 0.1 to more than 2 micron with a mean contour length of 0.6 to 0.8 micron, like that of covalently closed circular DNAs. Cells contained 400 to 1200 copies. The size distribution in the cytoplasts was narrow and the number-average length was 0.3 to 0.4 micron, whereas that in L6 karyoplasts was wide and the average length was 0.9 micron. The longer circular complexes appeared to be absent from the cytoplasts. The origin and biological functions of these complexes are discussed in relation to the cellular locations of the complexes.

Animals↗
PubMed1995

Mutational analysis of SpvR binding to DNA in the regulation of the Salmonella plasmid virulence operon.

The Salmonella plasmid-borne spvR gene encodes a 33-kDa regulatory protein that activates transcription of the spvABCD operon during the stationary phase of bacterial growth. We used gel mobility shift assays to demonstrate that SpvR recognizes a specific target DNA sequence within a 318-bp EcoRI-ApaI fragment upstream of spvA. The addition of unlabeled target DNA to the radioactive labeled DNA-SpvR complex resulted in competitive inhibition of band retardation confirming the specificity of SpvR binding. Introduction of target DNA on a high copy number plasmid into wild-type Salmonella dublin Lane resulted in a substantial decrease of SpvB synthesis, confirming the binding properties of this DNA segment in vivo. Three SpvR mutants were constructed and were shown to abolish the positive regulatory function of SpvR. By site-specific mutagenesis of spvR, three single amino acids within the putative SpvR N-terminal alpha-helix domains were substituted by prolines. This resulted in loss of binding to the spvA promoter sequence and in loss of activation of the spvABCD genes. This study demonstrates that the regulatory function of SpvR is mediated by specific binding to the promoter region of the spvABCD operon.

Amino Acid Sequence↗
PubMed1996

Structural heterogeneity of hsr(11) in the MDA-MB-134 mammary carcinoma cell line.

The MDA-MB-134 cell line was characterized by the presence of two homogeneously staining region (hsr) carrier chromosomes containing sequences from 8p11-p12, 11q13, and 8q24. Using fluorescence in situ hybridization, a detailed study of the organization of this chromosome has been performed. The hsr carrier chromosomes are shown to be identical and to derive from a chromosome 11, on which large segments of the long arm of chromosome 8 are translocated, forming its short arm. The hsr segment is inserted in the long arm, distally to C1NH gene. It is formed by sequences from both chromosomes 8 and 11. For the genes investigated by both methods the number of copies detected by in situ hybridization is compatible with that expected by quantification of Southern blots. In spite of its complexity, a possible mechanism of formation is proposed.

Breast Neoplasms↗
PubMedDate not supplied

Effects of occupational exposure to organic solvents upon cognitive performance.

Twenty-three individuals exposed to mixed organic solvents were compared with 23 nonexposed controls on a number of cognitive performance tasks. Solvent exposure resulted in a significantly poorer performance on the forward digit span test, copying of a complex figure, and on semantic memory tests which also measure individual's ability to integrate linguistic information into cohesive units. These tasks rely heavily upon short-term memory and its integrative operations in higher cognitive function. Acute exposure effect was also observed for the linguistic integrative task.

Adult↗
PubMedDate not supplied

Ammonium-mediated reduction of plasmid copy number and recombinant gene expression in Escherichia coli.

The effect of ammonium as a medium supplement on plasmid-encoded recombinant beta-galactosidase synthesis was explored in Escherichia coli cells during aerobic growth in complex medium. After induction, only doses of ammonium chloride below 1 g/L are able to transiently enhance the yield. However, the presence of nontoxic ammonium chloride concentrations of up to 10 g/L results in lower values of beta-galactosidase in a concentration-dependent fashion. A significant reduction in plasmid DNA content explains the decrease in the yield by a gene-dosage-involving mechanism.

Ammonium Chloride↗
PubMed1999

Tsessebe, Topi and Tiang: three distinct Tc1-like transposable elements in the malaria vector, Anopheles gambiae.

Three distinct types of Tc1-family transposable elements have been identified in the malaria vector, Anopheles gambiae. These three elements, named Tsessebe, Topi and Tiang, have the potential to encode transposases that retain most of the conserved amino acids that are characteristic of this transposon family. However, all three are diverged from each other by more than 50% at the nucleotide level. Full-length genomic clones of two types, Topi and Tsessebe, have been isolated and fully sequenced. The third, Tiang, is represented only by a 270 bp, PCR-amplified fragment of the transposase coding region. The Topi and Tsessebe elements are 1.4 kb and 2.0 kb in length, respectively, and differ in the length of their inverted terminal repeats (ITRs). The Topi elements have 26 bp ITRs, whereas the Tsessebe clones have long ITRs ranging in length from 105 to 209 bp, with the consensus being about 180 bp. This difference is due primarily to variation in the length of an internal stretch of GT repeats. The copy number and location of these elements in ovarian nurse cell polytene chromosomes varies greatly between element subtypes: Topi elements are found at between 17-31 sites, Tsessebe at 9-13 and Tiang at 20 euchromatic sites, in addition to several copies of these elements in heterochromatic DNA. The copy number and genomic insertion sites of these transposons varies between A. gambiae strains and between member species of the A. gambiae complex. This may be indicative of transpositionally active Tc1-like elements within the genome.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders.

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA 105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66 bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

Base Sequence↗