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Biomedical subjects

Laura L Stuve

Publications and source records attributed to Laura L Stuve.

7 recordsLinked to original sources
PubMed2005

Whole-genome patterns of common DNA variation in three human populations.

Individual differences in DNA sequence are the genetic basis of human variability. We have characterized whole-genome patterns of common human DNA variation by genotyping 1,586,383 single-nucleotide polymorphisms (SNPs) in 71 Americans of European, African, and Asian ancestry. Our results indicate that these SNPs capture most common genetic variation as a result of linkage disequilibrium, the correlation among common SNP alleles. We observe a strong correlation between extended regions of linkage disequilibrium and functional genomic elements. Our data provide a tool for exploring many questions that remain regarding the causal role of common human DNA variation in complex human traits and for investigating the nature of genetic variation within and between human populations.

Black or African American↗
PubMed2004

PCR isolation and cloning of novel splice variant mRNAs from known drug target genes.

Alternative splicing of pre-mRNAs is an important mechanism for the generation of vertebrate protein diversity. Unfortunately, the contribution of alternative splicing to protein diversity is currently not well understood because many full-length mRNA sequences have yet to be identified. In this report, we describe the use of RT-PCR to identify and clone 279 novel alternatively spliced mRNAs from 114 well-known drug target genes. Our findings demonstrate the existence of many novel alternatively spliced mRNA transcripts and suggest that many more genes undergo functionally significant alternative splicing than previously thought.

Alternative Splicing↗
PubMed2004

Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

Cloning, Molecular↗
PubMed2003

Cloning and characterization of a GABAA receptor gamma2 subunit variant.

We have cloned a novel gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit variant named gamma2XL. gamma2XL contains an alternatively spliced exon, resulting in the addition of 40 amino acids to the N-terminal extracellular domain between Ser171 and Tyr172. We show that gamma2XL failed to localize to the cell surface when it was coexpressed with the alpha2 and beta1 subunits in human embryonic kidney 293 cells. Expression of gamma2XL in 293 cells suppressed GABAA receptor binding in a dose-dependent manner by preventing GABAA receptor cell-surface localization. We also generated a gamma2 mutant with Ser171 and Tyr172 converted to glycine and threonine, respectively. We demonstrate that this mutant has a significantly lower affinity for the alpha2 and beta1 subunits and failed to reach the cell surface when coexpressed with these subunits. Together, our results indicate that Ser171 and Tyr172 in the gamma2 subunit constitute a critical motif. When this motif is disrupted by insertion of the alternative exon, access of the gamma2 subunit to the cell surface is prevented.

Alternative Splicing↗
PubMed2003

E. coli selection of human genes encoding secreted and membrane proteins based on cDNA fusions to a leaderless beta-lactamase reporter.

Although several signal peptide-trapping methods have been devised and used to detect signal sequences, none have relied on using E.coli to identify eukaryotic proteins with signal peptides. Here, we describe a system for selecting human secreted and membrane proteins in E. coli followed by the direct validation of secretion in human cells. The method is based on cDNA fusions to a leaderless beta-lactamase reporter gene to isolate clones encoding signal peptides of human genes. We found that beta-lactamase fusion proteins carrying a eukaryotic signal peptide at its N-terminus were able to direct their export into the periplasm in E. coli to confer survival upon challenge with carbenicillin. When libraries constructed from 5' end-enriched cDNAs fused to beta-lactamase were screened in E.coli, approximately 0.5%-1% of the cDNAs are selected, and over half of the surviving clones were found to encode for secreted fusion proteins when tested in human cells. These clones were sequenced and shown to represent human genes encoding signal peptides of secreted and membrane proteins. We conclude that this is an efficient and effective strategy to easily enrich cDNA libraries for the identification of novel genes likely to encode secreted enzymes, growth factors, and receptors.

Base Sequence↗
PubMed2003

Construction of cDNA libraries for highly efficient DNA sequencing from the 3' end of expressed genes.

The majority of expressed sequence tag (EST) sequences available today have been derived from the 5' ends of cDNA clones. Obtaining high-quality DNA sequences from the 3' ends of oligo(dT)-primed cDNA on a large scale has been difficult because of slippage of the DNA polymerase enzyme used in direct PCR and cycle sequencing. With the completion of whole genome sequencing for more and more organisms, mRNA 3'-UTR sequences can be particularly useful for clustering large numbers of ESTs for the effective discrimination of individual genes and gene families. We have identified a flaw in the widely used oligo(dT) primers for cDNA synthesis, and here we describe an improved priming approach to effectively synthesize cDNA devoid of homopolymeric nucleotide stretches from mRNA poly(A) tails to enable highly efficient and reliable DNA sequence determination from 3' mRNA ends. Using this method, we produced a rat lung cDNA library and successfully sequenced the 3' ends of 98% of all attempted clones.

Animals↗