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M A Batzer

Publications and source records attributed to M A Batzer.

At least 73 records · Page 4Linked to original sources
PubMed1994

A consensus Alu repeat probe for physical mapping.

Physical mapping of the human genome involves a variety of complex hybridization-based procedures, some of which rely upon the ability to separate human clones derived from human-rodent hybrid cell lines from those that contain background rodent-derived DNA sequences. The ability to block the repetitive element (Alu repeat) portion of inter-Alu PCR products derived from a variety of complex sources is also crucial for the isolation of unique DNA sequences. Here we report the construction and characterization of a new consensus Alu repeat probe (pPD39) designed for these purposes.

Animals↗
PubMed1994

A new bacteriophage P1-derived vector for the propagation of large human DNA fragments.

We have designed a P1 vector (pCYPAC-1) for the introduction of recombinant DNA into E. coli using electroporation procedures. The new cloning system, P1-derived artificial chromosomes (PACs), was used to establish an initial 15,000 clone library with an average insert size of 130-150 kilobase pairs (kb). No chimaerism has been observed in 34 clones, by fluorescence in situ hybridization. Similarly, no insert instability has been observed after extended culturing, for 20 clones. We conclude that the PAC cloning system will be useful in the mapping and detailed analysis of complex genomes.

Bacteriophage P1↗
PubMed1993

The use of polymorphic Alu insertions in human DNA fingerprinting.

We have characterized several Human Specific (HS) Alu insertions as either dimorphic (TPA25, PV92, APO), slightly dimorphic (C2N4 and C4N4) or monomorphic (C3N1, C4N6, C4N2, C4N5, C4N8) based on studies of Caucasian, Asian, American Black and African Black populations. Our approach is based upon: 1) PCR amplification using primers complementary to the unique DNA sequences that flank the site of insertion of the different Alu elements studied; 2) gel electrophoresis and scoring according to the presence or absence of an Alu insertion in one or both homologous chromosomes; 3) allele frequencies determined by gene counting and compared to Hardy-Weinberg expectations. Our DNA fingerprinting procedure using PCR amplification of diallelic polymorphic (dimorphic) Human Specific Alu insertions, may be used as a tool for genetic mapping, to characterize populations, study human migrational patterns, and track the inheritance of human genetic disorders.

DNA Fingerprinting↗
PubMed1993

Structural analysis and demonstration of the 29 kDa antigen of pathogenic Entamoeba histolytica as the major accessible free thiol-containing surface protein.

The 29 kDa protein of pathogenic Entamoeba histolytica is a cysteine-rich surface antigen which we recently characterized by cDNA sequencing and by using monoclonal antibodies which differentiated between pathogenic and non-pathogenic clinical isolates. To determine the structure and biochemical attributes of this protein, a repertoire of immunological techniques using monoclonal antibodies, and radiolabelling were employed. We demonstrated that the 29 kDa protein forms a 60 kDa dimer and a high-molecular-mass oligomer(s) on the surface of the organism through disulphide bonds, and is the major accessible free thiol-containing surface protein of E. histolytica. The deduced amino acid sequence encoding the 29 kDa protein was found to share a common amino acid domain with sequences reported for Helicobacter pylori, Salmonella typhimurium, MER5 gene expressed in murine erythroleukemia cells, Clostridium pasteurianum, and a Bacillus spp.

Amino Acid Sequence↗
PubMed1992

Master genes in mammalian repetitive DNA amplification.

The analysis of species-specific subfamilies of both the LINE and SINE mammalian repetitive DNA families suggests that such subfamilies have arisen by amplification of an extremely small group of 'master' genes. In contrast to the master genes, the vast majority of both SINEs and LINEs appear to behave like psudogenes in their inability to undergo extensive amplification.

Animals↗
PubMed1992

Molecular and cellular characterization of the 29-kilodalton peripheral membrane protein of Entamoeba histolytica: differentiation between pathogenic and nonpathogenic isolates.

To further characterize the 29-kDa surface antigen of Entamoeba histolytica, we analyzed the complete nucleotide sequence and compared the immunoreactivity of this antigen in pathogenic and nonpathogenic strains. Five cDNA clones (one 1.0-kb full-length clone, designated p13, and four partial-length clones) encoding the antigen were analyzed for allelic variation. Comparison of the nucleotide sequences revealed several single-nucleotide substitutions in all five cDNAs, two of which resulted in amino acid differences. Localization of the antigen to the amebic surface in a previous report (B. E. Torian, B. M. Flores, V. L. Stroeher, F. S. Hagen, and W. E. Stamm, Proc. Natl. Acad. Sci. USA 87:6358-6362, 1990) was corroborated by transmission electron microscopy showing colloidal gold particles on the surface of the trophozoites. Computer analysis of the deduced amino acid sequence predicted that the protein encoded by p13 was a hydrophilic peripheral membrane protein, and these data were confirmed by a Triton X-114 membrane extraction showing the presence of the 29-kDa antigen primarily in the aqueous phase of the detergent partition. Monoclonal antibodies to a fusion peptide differentiated between pathogenic and nonpathogenic clinical strains of E. histolytica in immunoblots. Although no immunoreactive epitopes were detected on nonpathogenic strains, Northern (RNA) analysis and DNA-DNA hybridization with a 700-bp cDNA probe demonstrated that mRNA and the gene encoding the 29-kDa surface antigen were present in both pathogenic and nonpathogenic clinical isolates.

Amino Acid Sequence↗
PubMed1992

Alu insertion polymorphism: a new type of marker for human population studies.

A PCR-based method was used to screen 462 individuals from Japan, Papua New Guinea, Indonesia, and Australia for an Alu family insertion polymorphism. The frequency of this Alu insertion shows significant heterogeneity among island subgroups of the Indonesian sample and between the Japanese-Indonesian populations and the Australian-New Guinean populations. The simple, rapid PCR-based screening technique and the significant frequency differences among populations demonstrate that Alu insertion polymorphisms are potentially valuable markers for studies of the evolutionary history and migration patterns of modern humans.

Australia↗
PubMed1991

Amplification dynamics of human-specific (HS) Alu family members.

We have investigated the distribution of several recently inserted Alu family members within representatives of diverse human groups. Human population studies using 65 unrelated human DNA samples, as well as a familial study to test inheritance, showed that individual Alu family members could be divided into three groups. The first group consisted of relatively older Alu family members which were monomorphic (homozygous) throughout the population tested (HS C3N1 and C4N6). The second group (HS C4N2, C4N5 and C4N8), apparently inserted into other repetitive regions of the genome, resulting in inconclusive results in the PCR test used. However, it is clear that these particular Alu insertions were present in a majority if not all of the loci tested. The third group was comprised of three dimorphic Alu family members (HS C2N4, C4N4 and TPA 25). Only a single Alu family member (TPA 25) displayed a high degree of dimorphism within the human population. This latter example also showed different allele frequencies in different human groups. The isolation and characterization of additional highly dimorphic Alu family members should provide a useful tool for human population genetics.

Alleles↗
PubMed1991

Evolution of the master Alu gene(s).

A comparison of Alu sequences that comprise more recently amplified Alu subfamilies was made. There are 18 individual diagnostic mutations associated with the different subfamilies. This analysis confirmed that the formation of each subfamily can be explained by the sequential accumulation of mutations relative to the previous subfamily. Polymerase chain reaction amplification of orthologous loci in several primate species allowed us to determine the time of insertion of Alu sequences in individual loci. These data suggest that the vast majority of Alu elements amplified at any given time comprised a single Alu subfamily. We find that, although the individual divergence relative to a consensus sequence correlate reasonably well with sequence age, the diagnostic mutations are a more accurate measure of the age of any individual Alu family member. Our data are consistent with a model in which all Alu family members have been made from a single master gene or from a series of sequential master genes. This master gene(s) accumulated diagnostic base changes, resulting in the amplification of different subfamilies from the master gene at different times in primate evolution. The changes in the master gene(s) probably occurred individually, but their appearance is clearly punctuated. Ten of them have occurred within an approximately 15-million-year time span, 40-25 million years ago, and 8 changes have occurred within the last 5 million years. Surprisingly, no changes appeared in the 20 million years separating these periods.

Animals↗
PubMed1991

A human-specific subfamily of Alu sequences.

Of a total of 500,000 Alu family members, approximately 500 are present as a human-specific (HS) subfamily. Each of the HS subfamily members shares a high degree of nucleotide identity and is not present at orthologous positions in other primate genomes, suggesting that HS subfamily members have recently inserted within the human genome. This confirms the hypothesis that the majority of Alu family members are amplified copies of a "master" gene(s). This master gene appears to be amplifying at a rate much slower than that seen earlier in primate evolution. Some of the HS Alu subfamily members have amplified so recently that they are dimorphic in the human population, making them a potentially powerful tool for studies of human populations.

Animals↗
PubMed1990

Structure and variability of recently inserted Alu family members.

The HS subfamily of Alu sequences is comprised of a group of nearly identical members. Individual subfamily members share 97.7% nucleotide identity with each other and 98.9% nucleotide identity with the HS consensus sequence. Individual subfamily members are on the average 2.8 million years old, and were probably derived from a single source 'master' gene sometime after the human/great ape divergence. The recent Alu family member insertions provide a better image of the structure of Alu retroposons before they have had the opportunity to change significantly. All of the HS subfamily members are flanked by perfect direct repeats as a result of insertion at staggered nicks. The 'master' gene from which the HS subfamily members were derived had an oligo-dA rich tail at least 40 bases long. The 'master' gene is very rich in CpG dinucleotides, but nucleotide substitutions within subfamily members accumulated in a random manner typical for Alu sequence with CpG substitutions occurring 9.2 fold faster than non-CpG substitutions.

Base Sequence↗
PubMed1988

Molecular analysis of alcohol dehydrogenase electromorphs in wild type and transformed Drosophila melanogaster.

The protein expressed by the alcohol dehydrogenase locus (Adh) in D. melanogaster comprises a small group of electromorphs. We are able to study the expression of these electromorphs by electrophoretic separation and subsequent probing of blots of the separated polypeptides with antiserum for alcohol dehydrogenase (ADH). In the present study we have utilized this technique to study and compare the ADH electromorphs in wild type D. melanogaster with D. melanogaster transformants which carry an Adh gene from D. grimshawi, D. hawaiiensis or D. affinidisjuncta and produced functional ADH (10, 19). We have determined that polypeptides are produced by the donor loci in the transformed flies and further show that although the molecular weight of the expressed polypeptides is similar to D. melanogaster electromorphs, the isoelectric points are not similar. Thus this methodology offers the potential to study naturally occurring ADH electromorphs and null alleles independent of enzymatic activity assays.

Alcohol Dehydrogenase↗
PubMed1988

Spectra of molecular changes induced in DNA of Drosophila spermatozoa by 1-ethyl-1-nitrosourea and X-rays.

Mutations induced in Drosophila spermatozoa at the alcohol dehydrogenase Adh locus by 1-ethyl-1-nitrosourea (ENU) were compared to X-ray-induced mutations using genetic tests for complementation, southern blotting, western blotting and northern blotting. 8 of 10 ENU-induced mutations complemented all known adjacent loci and were presumed to be intragenic. In contrast, 8 of 30 X-ray-induced mutations were intragenic. Southern blot analysis showed that 2 of 7 intragenic mutations induced by X-rays were altered at the Adh locus, whereas all 8 intragenic ENU mutants appeared normal. Western blot analysis showed 4 of 7 intragenic mutants induced by X-rays produced a detectable polypeptide; 1 of the 4 had normal molecular weight and charge. In contrast, 7 of the 8 intragenic mutants induced by ENU produced a polypeptide of normal molecular weight and charge. One ENU and two X-ray-induced mutants, which had normal southern blots and no detectable polypeptide, produced normal molecular weight mRNA by northern blots. The interpretation of these results is that in spermatozoa X-rays induce primarily deletions that either produce deficiencies of the Adh locus or nonsense mutations within the locus, whereas ENU induces primarily missense mutations. This forward mutation assay based on loss of enzymatic activity efficiently recovered a broad spectrum of mutations ranging from missense to intragenic deletions and multi-locus deficiencies. Only 3 of these 40 mutations produced a polypeptide detectable as an electrophoretic variant.

Alcohol Dehydrogenase↗
PubMed1988

Molecular analysis of Drosophila melanogaster AdhnLA405 confirms reliability of DNA-sequencing methodology.

An alcohol dehydrogenase (ADH) null mutant of Drosophila melanogaster (AdhnLA405) originally recovered following X-ray irradiation of mature sperm (Aaron, 979) is analyzed by Southern blotting, Western blotting, and DNA sequencing. The genetic, immunologic, and nucleic acid sequence data are consistent with the hypothesis that a cross-over event, independent of X-irradiation, between parental chromosomes is responsible for the ADH null phenotype of AdhnLA405. By DNA-sequence analysis we show that molecular cloning of this locus (i.e., propagation in prokaryotic hosts) apparently does not introduce any spurious changes (substitutions, additions, deletions, or rearrangements) within the DNA.

Alcohol Dehydrogenase↗
PubMed1986

An in vitro screening system for the nephrotoxicity of various platinum coordination complexes. A cytochemical study.

Isolated rat kidney tubules were cultured in Earle's medium with and without the platinum coordination complexes. Aliquots were taken at 0, 1, 2, 3, 4, 5, 6, and 8 h and analyzed for the amount of Na+/K+-ATPase, Ca2+-ATPase, alkaline phosphatase, and acid phosphatase. Culture medium was also analyzed biochemically for the amounts of alkaline phosphatase present. There is a decrease in the various enzymes levels of the tubules after incubation in nephrotoxic analogues with a corresponding increase in the culture medium. These results compare favorably with in vivo studies. The alkaline phosphatase monitored in the rat kidney cross sections from both the normal and the drug-treated animals at 0, 3, 5, 10, and 20 days showed a correlation in the decrease of enzyme levels in the kidney with a corresponding increase in the urinary levels in both the Wistar and the Long Evans rats. The baseline levels were higher in the Long Evans rats than in the Wistar rats. After cisplatin (nephrotoxic) treatment the Long Evans rats had twice as much alkaline phosphatase in the urine at day 5 as the Wistar rats. Rats treated with cyclobutanedicarboxylatoplatinum (II) did have some alkaline phosphatase output in the urine in excess of the normal levels, but this increase was not so highly significant as to justify classifying the drug as nephrotoxic.

Acid Phosphatase↗