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M Engelstein

Publications and source records attributed to M Engelstein.

17 recordsLinked to original sources
PubMed2002

High-speed plasmid isolation using 96-well, size-exclusion filter plates.

The accelerating pace of genomics analysis has necessitated the abbreviation of DNA sample preparation protocols. We have developed a size-exclusion-based system for the rapid isolation ofplasmid DNA in a 96-well microplate format. This high-speed protocol employs a modified alkaline lysis methodfor the preparation of the bacterial lysate, followed by three short vacuum filtration steps. Unlike traditional bind/wash/elute methods, there is no need to use chaotropic salts or ethanol. The samples are recovered from the top side of the MultiScreen96 PLASMID plates. Starting with bacterial cell pellets, the entire prycedure for purifying the plasmid DNA can be performed in 30 min with a multichannel pipettor. The high yields, reproducibility, and quality of the plasmids make this system a good choice for any cloning or DNA sequencing operation.

Base Sequence↗
PubMed1998

An efficient, automatable template preparation for high throughput sequencing.

We have developed a 96-well format for DNA template isolation that can be readily automatable. The template isolation protocol involves simple alkaline lysis chemistry and reversible capture on a silica solid phase. After the cells are lysed, no centrifugation is necessary, as lysate purification, DNA binding, washing, and release occur in 96-well filter plates. Large numbers of templates prepared using the silica purification method have been sequenced and analyzed. The quality of sequence resulting from our method has been compared with that generated from several commercial plasmid preparation protocols. We found sequence quality of the silica bead preparations to be equivalent to or, in some cases, better than those prepared by other methods. This method offers many advantages over other protocols we have used. First, the silica purifications have allowed us to more than double overall laboratory throughput while decreasing our template isolation materials cost at least five-fold. Second, because we have eliminated all centrifugation steps in the protocol, automation has been much simpler. The protocol has also been adapted to purify PCR products for use as templates in subsequent sequencing reactions.

Automation↗
PubMed1997

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome.

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of the carboxy-terminal portion of p53 using both yeast and mammalian cell assays in vivo.

Increasing evidence indicates that p53 is a transcriptional trans-activator through its sequence-specific DNA binding domain. Tumor-derived p53 mutations disrupt the trans-activation ability mainly due to loss of its sequence-specific DNA binding. Using both yeast and mammalian cell assays, the effect of p53 mutations in the carboxy terminal portion was investigated in order to address how p53 mutations outside of the DNA binding domain affect p53 function. The p53 cDNA in the carboxy-terminus was randomly mutagenized by error-prone polymerase chain reactions and the amplified cDNA was screened for the ability to trans-activate using a yeast assay. Four p53 mutations, including two missense and two nonsense mutations located in the carboxy-terminal oligomerization domain, were further analysed for trans-activation, cell cycle arrest and colony formation in a human osteosarcoma cell line, Saos-2. These functional properties of p53 were disrupted by the missense mutations. Surprisingly, one of the nonsense mutations disrupts the trans-activation function and the ability to G1 arrest but shows a strong inhibition of colony formation. These results confirm that mutations in the oligomerization domain can inactivate p53 function and also indicate that p53-mediated cell growth inhibition does not necessarily depend on the ability to arrest cell cycle.

Amino Acid Sequence↗
PubMed1995

A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene.

The GTPase-activating protein related domain of the human neurofibromatosis type 1 protein (NF1GRD) can down-regulate RAS in Saccharomyces cerevisiae. Using a technique termed the FASAY method, for Functional Analysis of Separated Alleles in Yeast, we designed a rapid method for detection of heterozygous NF1GRD loss-of-function mutations. In our method, PCR amplified NF1GRD cDNA is directly cloned into a centromeric vector by homologous recombination in a cdc25 temperature-sensitive mutant strain expressing human Ha-ras. This strain is dependent on the Ha-ras for growth, allowing a simple growth assay for NF1GRD loss-of-function mutations. In a test of our method, two alternatively spliced NF1GRD cDNAs (type I and II) inhibited yeast growth whereas four mutants with amino acid substitutions at highly conserved residues did not. This simple method thus permits the rapid screening for heterozygous germline or somatic NF1GRD mutations. In an initial application of this method, no mutations disrupting NF1GRD function were detected in lymphoblasts from 11 previously untested neurofibromatosis type 1 patients.

Base Sequence↗
PubMed1994

Characterization of human Gadd45, a p53-regulated protein.

GADD45 (growth arrest and DNA damage) is a DNA-damage-inducible gene regulated in part by the tumor suppressor p53. A role in negative growth control has recently been suggested based on significant (more than 75%) reduction of colony formation following over expression of Gadd45. To better understand the role of Gadd45, we have developed specific rabbit and murine antibodies raised against the human recombinant protein. Using these antibodies, we have found that in ML-1 cells Gadd45 is predominantly a nuclear protein. MyD118, a protein induced by terminal differentiation sharing 57% homology with Gadd45, does not cross-react with any of the antibodies produced. As expected, the induction of Gadd45 protein by ionizing radiation (IR) was also found to be dependent on a wild type p53 phenotype. Interestingly, WI-L2-NS, a human lymphoid cell line, showed very high basal levels of Gadd45 mRNA and protein in addition to a high constitutive level of a mutated p53 protein. In this cell line, the high levels of GADD45 did not inhibit cellular growth in spite of the fact that no mutations were found in GADD45 sequence. These results indicate that some cell line(s) can tolerate high levels of Gadd45 and abrogate its growth suppression function.

Animals↗
PubMed1994

Linkage of cutaneous malignant melanoma/dysplastic nevi to chromosome 9p, and evidence for genetic heterogeneity.

We examined the relationship between cutaneous malignant melanoma/dysplastic nevi (CMM/DN) and chromosome 9p in 13 pedigrees with two or more living cases of invasive melanoma. We used two highly informative (CA)n repeats, D9S126 and IFNA, previously implicated in familial malignant melanoma (MLM), to conduct linkage analysis. Three analyses were performed: (1) CMM alone--all individuals without either confirmed melanoma or borderline lesions were considered unaffected (model A); (2) CMM/DN with both variable age at onset and sporadics (model B); and (3) CMM affecteds only--all individuals either without confirmed melanoma or with borderline lesions were designated "unknown" (model C). There was significant evidence for linkage to IFNA in all three models. For CMM alone, the maximum lod score (Zmax) was 4.36 at theta = .10 for model A and 3.39 at theta = .10 for model C. For CMM/DN (model B), Zmax = 3.05 at theta = .20. There was no significant evidence for linkage between CMM alone or CMM/DN and chromosome 9p marker D9S126. In addition, there was significant evidence for heterogeneity when a homogeneity test allowing for linkage to chromosome 9p or chromosome 1p or neither region was used. These results suggest that there is an MLM susceptibility locus on chromosome 9p but that familial melanoma is heterogeneous and not all families with CMM/DN are linked to a locus in this region.

Adolescent↗
PubMed1993

A PCR-based linkage map of human chromosome 1.

A genetic linkage map of human chromosome 1 based entirely on PCR-typable markers has been developed using 38 simple sequence repeat (SSR) polymorphisms. These SSRs include 36 dinucleotide repeats and 2 tetranucleotide repeats. The average heterozygosity at these markers was 0.73 and ranged from 0.52 to 0.95. Multipoint linkage analysis was used to develop a map of these 38 markers in which the relative placement of each locus is supported by likelihood odds > 1000:1. This PCR-based map was anchored at the centromere by the D1Z5 alpha-satellite polymorphism, and the ends of the map were defined by D1Z2 and D1S68, which are the most distal loci in the CEPH consortium map of chromosome 1. The sex-averaged, male, and female maps extend for 328, 273, and 409 cM, respectively. The average distance between markers on the sex-averaged map is 8 cM, and the largest interval is 32 cM. This map of highly informative PCR-based markers will provide a rapid means of screening human chromosome 1 for the presence of disease genes.

Base Sequence↗
PubMed1992

Isolation and chromosomal assignment of 100 highly informative human simple sequence repeat polymorphisms.

One hundred highly informative simple sequence repeat (SSR) polymorphisms have been isolated and mapped to specific human chromosomes by somatic cell hybrid analysis. These markers include 97 (CA)n, 2 (AGAT)n, and a single (AACT)n repeat. All the SSRs have heterozygosities greater than 0.50 and can be amplified using identical PCR conditions. At least one SSR was detected on every chromosome, except for chromosomes 22 and Y. The frequency of (CA)n repeats on each chromosome was proportional to the relative chromosomal length, except for chromosome 15, on which a substantial excess of markers was identified.

Base Sequence↗
PubMed1992

Genetic and developmental analysis of polytene section 17 of the X chromosome of Drosophila melanogaster.

Polytene section 17 of the X chromosome of Drosophila melanogaster, previously known to contain six putative lethal complementation groups important in oogenesis and embryogenesis, has here been further characterized genetically and developmentally. We constructed fcl+Y, a duplication of this region, which allowed us to conduct mutagenesis screens specific for the region and to perform complementation analyses (previously not possible). We recovered 67 new lethal mutations which defined 15 complementation groups within Df(1)N19 which deletes most of polytene section 17. The zygotic lethal phenotypes of these and preexisting mutations within polytene section 17 were examined, and their maternal requirements were analysed in homozygous germline clones using the dominant female sterile technique. We present evidence that an additional gene, which produces two developmentally regulated transcripts, is located in this region and is involved in embryogenesis, although no mutations in this gene were identified. In this interval of 37 to 43 polytene chromosome bands we have defined 17 genes, 12 (71%) of which are of significance to oogenesis or embryogenesis.

Amino Acid Sequence↗
PubMed1990

Activation of polyubiquitin gene expression during developmentally programmed cell death.

Ubiquitin, a highly conserved 76 amino acid protein, plays a role in targeting intracellular proteins for degradation. Ubiquitin expression was examined during the developmentally programmed atrophy and degeneration of the intersegmental muscles (ISMs) in the hawk-moth, Manduca sexta. A clone containing nine repeats of the ubiquitin coding sequence was isolated from an ISM cDNA library and was used as a probe to examine polyubiquitin expression during development. When the ISMs became committed to degenerate, polyubiquitin gene expression increased dramatically. Injection of 20-hydroxyecdysone, which delays degeneration in this system, prevented the increase in polyubiquitin mRNA. The expression of polyubiquitin occurred without apparent activation of the cell's heat shock response. These data suggest that ubiquitin plays a role in programmed cell death.

Animals↗