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Biomedical subjects

E M Golenberg

Publications and source records attributed to E M Golenberg.

9 recordsLinked to original sources
PubMed2002

Genetic diversity and response to IFN of the NS3 protease gene from clinical strains of the hepatitis C virus.

The N-terminal one-third of the hepatitis C virus nonstructural gene 3 (NS3) codes for a serine protease. To investigate natural genetic diversity of this enzyme a nested PCR reaction was developed to obtain NS3 protease sequence data directly from patient strains. This data was used to determine genetic diversity, phylogenetic and evolutionary rates, and selection of variants by interferon therapy. The potential effect of genetic diversity on enzyme structure using molecular modeling was also attempted. Results show significant variability in clinical HCV strains at both the nucleotide (30.2% for 1a and 25.8% for 1b) and amino acid sequences (11.0% for 1a and 9.9% for 1b). Phylogenic analysis shows two distinct clades with two HCV isolates grouping as a sister clade to 1b. Structural analysis reveals that most mutations lie in the N-terminus of the enzyme. When strains were sorted as to whether or not the patient had received antiviral therapy, no difference was found in the number or locations of mutations in 1a strains. However, 1b strains demonstrated an overall drop in the number of positions that were mutated. This study demonstrates significant differences among natural strains that may pose a problem for structure based drug development.

Adult↗
PubMed1997

A blind testing design for authenticating ancient DNA sequences.

Reproducibility is a serious concern among researchers of ancient DNA. We designed a blind testing procedure to evaluate laboratory accuracy and authenticity of ancient DNA obtained from closely related extant and extinct species. Soft tissue and bones of fossil and contemporary museum proboscideans were collected and identified based on morphology by one researcher, and other researchers carried out DNA testing on the samples, which were assigned anonymous numbers. DNA extracted using three principal isolation methods served as template in PCR amplifications of a segment of the cytochrome b gene (mitochondrial genome), and the PCR product was directly sequenced and analyzed. The results show that such a blind testing design performed in one laboratory, when coupled with phylogenetic analysis, can nonarbitrarily test the consistency and reliability of ancient DNA results. Such reproducible results obtained from the blind testing can increase confidence in the authenticity of ancient sequences obtained from postmortem specimens and avoid bias in phylogenetic analysis. A blind testing design may be applicable as an alternative to confirm ancient DNA results in one laboratory when independent testing by two laboratories is not available.

Animals↗
PubMed1997

Proboscidean DNA from museum and fossil specimens: an assessment of ancient DNA extraction and amplification techniques.

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.

Animals↗
PubMed1996

Effect of highly fragmented DNA on PCR.

We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.

DNA↗
PubMed1996

Phylogenetic resolution within the Elephantidae using fossil DNA sequence from the American mastodon (Mammut americanum) as an outgroup.

DNA was extracted from the extinct American mastodon, the extinct woolly mammoth, and the modern Asian and African elephants to test the traditional morphologically based phylogeny within Elephantidae. Phylogenetic analyses of the aligned sequences of the mitochondrial gene cytochrome b support a monophyletic Asian elephant-woolly mammoth clade when the American mastodon is used as an outgroup. Previous molecular studies were unable to resolve the relationships of the woolly mammoth, Asian elephant, and African elephant because the sequences appear to have evolved at heterogeneous rates and inappropriate outgroups were used for analysis. The results demonstrate the usefulness of fossil molecular data from appropriate sister taxa for resolving phylogenies of highly derived or early radiating lineages.

Africa↗
PubMed1993

Evolution of a noncoding region of the chloroplast genome.

The relative rate of occurrence of nucleotide substitutions versus indel (insertion/deletion) events is investigated by comparing complete DNA sequence data from the noncoding portion of the chloroplast genome that maps between the genes rbcL and atp beta. The sequence data are obtained from nine species that represent three tribes of the grass family. Indels could be categorized by those that are deletions or duplications of adjacent or proximal sequences and those that do not appear to be permutations of adjacent sequences. The first category represents 82% of the recorded indels. These indels may also be characterized by being direct duplications of one to several bases usually within runs of As or Ts or by being duplications or deletions of more complex sequences. When viewed from within groups of closely related taxa, indel events appear to occur at an equal or slightly faster rate than do nucleotide substitution events. However, the apparent rate of accumulation of indels in more distantly related species is significantly slower than that of nucleotide substitutions. This difference in apparent accumulation rates between indel events and nucleotide substitutions suggests that the proportion of superimposed changes has been higher among all indel events than among all nucleotide substitution events. Indeed the indels involving more complex sequences were found to be confined across taxa to a number of highly labile sites. Independent, though similar, indel events occur at identical sites in unrelated taxa, yet may not be shared among related taxa, resulting in a type of molecular parallelism. As a result, the phylogenetic tree based on indel events represents an evolutionary hypothesis which is inconsistent with the accepted phylogeny of these grasses. The phylogenetic tree based on nucleotide substitutions is consistent with accepted phylogeny.

Base Sequence↗
PubMed1991

Amplification and analysis of Miocene plant fossil DNA.

Ancient DNA has been extracted and sequenced from several animal and plant specimens. Previous considerations of the damage to ancient DNA have suggested that both the age and size of DNA fragments that can be retrieved and sequenced may be limited, the former to between several thousand and at most tens of thousands of years old, and the latter to at most a few hundred bases. A recent report of a 770 base pair (b.p.) sequence from the chloroplast gene rbcL from a Miocene Magnolia latahensis leaf indicates that both estimated limitations may be too conservative. Further work has indicated that analysis of Miocene fossil DNA can be replicated, and can, therefore, open up the prospects for future development of the field of molecular palaeontology. Successful amplification of fossil DNA is sometimes confounded by factors inherent to fossil DNA or to samples with minimal amounts of target DNA. Techniques that alter denaturation, reduce inhibitors and the problem of contaminants, and repair DNA prior to polymerase chain reaction amplification can increase the probability of success.

Base Sequence↗
PubMed1990

Chloroplast DNA sequence from a miocene Magnolia species.

DNA has been successfully extracted from several samples of preserved tissue, the oldest so far reported originating from a 13,000-year-old ground sloth. Both severe damage to the preserved DNA, primarily due to oxidation of the pyrimidines, has prevented the acquisition of sequence data from ancient samples except in a few cases. We report here the extraction of DNA from fossil leaf samples from the Miocene Clarkia deposit (17-20 Myr old), the amplification of an 820-base pair (bp) DNA fragment from the chloroplast gene rbcL from a fossil of the genus Magnolia, and its subsequent sequencing. The sequence was verified by comparison with published and unpublished rbcL sequences. These results extend our ability to analyse ancient DNA and may open new avenues into problems in palaeobotany, biogeography, and in the calibration of mutation rates.

Base Sequence↗