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M Bittner

Publications and source records attributed to M Bittner.

54 records · Page 3Linked to original sources
PubMed1997

Use of microgenomic technology for analysis of alterations in DNA copy number and gene expression in malignant melanoma.

Chromosome abnormalities in human malignancies have identified the genomic location of several important growth-regulatory genes, including cellular oncogenes and tumour suppressor genes. Melanomas are characterized by recurring chromosome alterations, and it is important to identify those genes whose altered expression may be causally related to melanocytic transformation. This short report presents an overview of strategies used which combine the materials and technologies of the Human Genome Project with clinically directed studies of melanoma biology. The Human Genome Project combines various technologies, including cytogenetic, physical mapping, genetic mapping and DNA sequencing, in order to identify all of the human genes, but especially the 4000 estimated to contribute to human disease. This report focuses first on advances in genome technology that provide information on chromosome rearrangements and DNA copy number changes. This includes a discussion of chromosome microdissection as well as the microexcision of tissue specimens to gain insights into chromosome regions altered in association with melanocyte transformation. Next, there is a brief discussion of the generation and characterization of subtracted cDNA sublibraries which allow the identification of genes uniquely expressed in association with the transformed phenotype of human melanoma cells. Finally, we briefly discuss the feasibility of using a recently developed system for parallel examination of multiple genes based upon robotic printing of cDNAs on glass slides, and simultaneous two-colour fluorescence hybridization to study the expression patterns of cDNAs for their association with melanoma tumour suppression. The combination of these varied molecular technologies may provide insights into previously unrecognized genes involved causally in the pathobiology of this important neoplasm, and may provide new targets for clinical intervention.

Chromosome Mapping↗
PubMed1996

Molecular cytogenetic characterization and physical mapping of 12q13-15 amplification in human cancers.

Amplification of sequences derived from 12q13-15 is frequent in human sarcomas and brain tumors. Detailed mapping studies of the amplified region are necessary for definition of the impact of these amplification events on the tumor cell phenotype. By using the genes in this region and genomic fragments isolated by chromosome microdissection, we have established a series of ordered probes from 12q13-15 for fluorescence in situ hybridization (FISH) and Southern blot analysis. These probes have been used for physical mapping of two portions of the interval from GLI to D12S8. The centromeric region extends 1.8 Mb from GLI to microclone M79 and contains at least five genes, including the cyclin-dependent kinase gene CDK4. The more telomeric region includes the p53 regulator MDM2 and covers 1.1 Mb. We used the same group of probes to determine the pattern of amplification in three cell lines and three tumor specimens carrying amplified sequences from 12q13-15. In addition, we used a yeast artificial chromosome (YAC) contig of several megabases covering the entire region from SAS to D12S8 for FISH to determine the pattern of amplification in the neuroblastoma cell line NGP-127. The results suggest that the MDM2 and CDK4 regions may be either coamplified or amplified independently, and they illustrate how the map positions of genes and their functions may interact to determine the pattern of DNA amplification in human malignancies.

Base Sequence↗
PubMed1995

Isolation of YAC insert sequences by representational difference analysis.

We present a method for the isolation of YAC insert sequences by representational difference analysis (RDA). To achieve maximal representation of the sequences, the amplicons were generated from a Mbol digestion product. RDA was performed using a 970 kb insert YAC clone. After two rounds of re-association and selective amplification 92% of the difference product represented sequences derived from the YAC insert. Twenty insert-specific sequence-tagged sites were readily defined. The difference product was also successfully used to isolate microsatellite markers, to identify clones from a human PAC library and as a chromosome painting probe in fluorescence in situ hybridization.

Base Sequence↗
PubMed1995

Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.

Homozygous deletions have been central to the discovery of several tumor-suppressor genes, but their finding has often been either serendipitous or the result of a directed search. A recently described technique [Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, 946-951] held out the potential to efficiently discover such events in an unbiased manner. Here we present the application of the representational difference analysis (RDA) to the study of cancer. We cloned two DNA fragments that identified a homozygous deletion in a human pancreatic adenocarcinoma, mapping to a 1-centimorgan region at chromosome 13q12.3 flanked by the markers D13S171 and D13S260. Interestingly, this lies within the 6-centimorgan region recently identified as the BRCA2 locus of heritable breast cancer susceptibility. This suggests that the same gene may be involved in multiple tumor types and that its function is that of a tumor suppressor rather than that of a dominant oncogene.

Adenocarcinoma↗
PubMed1995

Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analog and tissue-specific expression suggest a role in sink-source regulation.

Photoautotrophic suspension-culture cells of Chenopodium rubrum that were shifted to mixotrophic growth by adding glucose were used as model system to investigate the influence of the source-sink transition in higher plants on the expression and enzyme activities of intracellular and extracellular invertases. The complete cDNA coding for an extracellular invertase was cloned and sequenced from C. rubrum, and its identity has been proven by heterologous expression in Saccharomyces cerevisiae. The higher activity of extracellular invertase after preincubation in the presence of glucose was paralleled by an increased expression of the corresponding gene. The induction by glucose could be mimicked by the nonmetabolizable glucose analog 6-deoxyglucose. Both enzyme activity and mRNA level of extracellular invertase showed a sink-tissue-specific distribution in plants. The activity of neutral and acidic intracellular invertases were not affected by preincubation of autotrophic tissue cultures with sugars, nor did they show a tissue-specific distribution in plants. The data suggest that apoplastic invertase not only has an important function in phloem unloading and carbohydrate partitioning between source and sink tissues but may also have a role in establishing metabolic sinks.

Amino Acid Sequence↗
PubMed1984

Site-specific cleavage of DNA at 8- and 10-base-pair sequences.

A method is described for cutting DNA at specific sites that are 8 and 10 base pairs long. The DNA is first treated with a specific methylase, either the restriction-modification enzyme M. Taq I, which converts the 4-base sequence T-C-G-A to T-C-G-mA, or the similar enzyme M. Cla I, which converts the 6-base sequence A-T-C-G-A-T to A-T-C-G-mA-T. The DNA is then cleaved with Dpn I, a restriction endonuclease that recognizes the sequence G-mA-T-C. Dpn I is unique in that it cuts only DNA that is methylated at adenine in both strands of its recognition sequence. In DNAs that are not otherwise methylated at adenine in both strands of the sequence G-A-T-C, cleavage by Dpn I occurs only at the following sequences: in the case of M. Taq I methylation, 5' T-C-G-mA - T-C-G-mA 3' 3' mA-G-C - T-mA-G-C - T 5'; in the case of M. Cla I methylation, 5' A - T-C-G-mA - T-C-G-mA-T 3' 3' T-mA-G-C - T-mA-G-C - T-A 5'. Specific cutting and cloning at these methylase/Dpn I-generated sites is shown experimentally. Further, we describe how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs. In DNA that contains equal amounts of each base distributed at random, 8- and 10-base-pair recognition sequences occur, on the average, approximately once every 65,000 and 1,000,000 base pairs, respectively. Potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.

Base Composition↗
PubMed1982

Cloning vectors derived from bacterial plasmids.

A wide variety of plasmid cloning vectors, most of which utilize the basic Co1E1I replicon have been constructed. Utilizing these vectors, in conjunction with the newly developed techniques of gene isolation and oligonucleotide synthesis, essentially any gene which can be identified can be cloned. We anticipate that future work in this area will be directed at improving techniques for the regulated expression of cloned genes and the further development of plasmid replicons in which the copy number can be readily controlled.

Bacteriophage lambda↗
PubMed1981

Versatile cloning vectors derived from the runaway-replication plasmid pKN402.

Two cloning vectors have been constructed employing runaway-replication mutants of plasmid R1. One of these, pMOB45, carries tetracycline and chloramphenicol resistance. The other, pMOB48, carries chloramphenicol resistance, lacOP, and an assayable part of the lacPOZ operon. Both of these plasmids can be amplified to high levels by heat induction, which condition does not lead to inhibition of protein synthesis; thus the plasmid can produce large amounts of DNA and protein. In pMOB48, a unique BamHI site is present near the amino-terminus of the beta-galactosidase gene. Chimeras formed by the insertion of restriction fragments at this site can be detected on X-gal plates, and can be used for the lacIq-controlled expression of proteins which are fused to the amino-terminus of beta-galactosidase. Induction with IPTG at 40 degrees C leads to the synthesis of extremely high levels of proteins whose gene have been cloned into this site.

Chloramphenicol↗
PubMed1981

Direct identification of sickle cell anemia by blot hybridization.

Several reports have been published on the use of polymorphisms found in the human hemoglobin genes as a means for prenatal diagnosis of sickle cell anemia. The disadvantages of this approach reside in its limited application and the need for family analysis. Here we report that, by use of restriction endonuclease Dde I and diazobenzyloxymethyl-paper transfer procedures, a direct analysis can be made. Individuals with normal hemoglobin (AA) show two bands (175 and 201 base pairs) complementary to a 5'-specific beta-globin gene probe. Sickle cell trait individuals (AS) exhibit an additional band (376 base pairs). Individuals with sickle cell anemia (SS) show the band at 376 base pairs with a concomitant loss of the 175-base pair band. We interpret these changes in banding pattern to be the result of the elimination of a restriction site for Dde I in the altered codon associated with the sickle cell allele. Because an analysis can be performed on as little as 20 micrograms of cellular DNA, the application to prenatal diagnosis of sickle cell anemia should be possible.

Anemia, Sickle Cell↗
PubMed1979

Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities.

Detailed procedures are presented which allow reproducible preparation of T4 gene 32 protein, a helix-destabilizing protein essential for DNA replication and genetic recombination in T4 bacteriophage-infected Escherichia coli cells. Although 32 protein can be purified to better than 99% homogeneity by any one of several procedures, these methods have been developed to remove trace amounts of contaminating deoxyribonucleases, which are present in high levels in the original infected cells. Two alternative preparations are presented, each involving three chromatographic steps. Both 32 proteins obtained are essentially "nuclease-free," when tested at physiological salt concentrations. However, we show here that the phenyl-Sepharose chromatography step, which is necessary to remove an exonuclease activity active only at low salt concentrations, also removes a second protein present in trace amounts. In some cases, retention of this second protein is desirable, since it is essential for obtaining RNA primed, de novo DNA chain starts in an in vitro DNA replication system, when this system is constructed by mixing highly purified preparations of each of the six replication proteins coded for by T4 genes 32, 43, 44, 62, 45, and 41.

DNA Helicases↗
PubMed1979

The effects of methyl-3-O-methyl gallate on isolated nerve activity.

Methyl-3-O-methyl gallate (M30MG), extracted from Crinodendron hookerianum Gay, decreased both sciatic nerve accommodation and the amplitude of the compound action potential and increased the threshold to rectangular and exponential currents applied to the isolated sciatic nerve in a manner similar to that observed when tetrodotoxin is applied to the nerve. M30MG would act mainly on the large nerve fibres by a mechanism probably resembling that of tetrodotoxin.

Animals↗
PubMedDate not supplied

Screening of Chilean plants for anticancer activity. I.

Extracts prepared from 519 samples of plant material were evaluated for anticancer activity against the usual systems. The results of the antitumor testing showed that 156 of these extracts gave initial indications of anticancer activity in one or more tumor systems. The activity was confirmed in 14 samples.

Antineoplastic Agents, Phytogenic↗