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D W Russell

Publications and source records attributed to D W Russell.

At least 163 records · Page 9Linked to original sources
PubMed1986

Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit.

The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal with familial hypercholesterolemia, produces a mutant receptor for plasma low-density lipoprotein (LDL) that is not transported to the cell surface at a normal rate. Cloning and sequencing of complementary DNA's from normal and WHHL rabbits, shows that this defect arises from an in-frame deletion of 12 nucleotides that eliminates four amino acids from the cysteine-rich ligand binding domain of the LDL receptor. A similar mutation, detected by S1 nuclease mapping of LDL receptor messenger RNA, occurred in a patient with familial hypercholesterolemia whose receptor also fails to be transported to the cell surface. These findings suggest that animal cells may have fail-safe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues.

Amino Acid Sequence↗
PubMed1986

The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors.

Genomic DNA encompassing the terminal exons of the gene for the low density lipoprotein (LDL) receptor was isolated from J.D., a patient with familial hypercholesterolemia whose receptor fails to cluster in coated pits. The DNA sequence revealed a substitution of a cysteine codon for a tyrosine codon at residue 807 in the cytoplasmic domain of the receptor. We reproduced this substitution through oligonucleotide-directed mutagenesis of the normal human receptor cDNA. Upon transfection into receptor-deficient hamster cells, the cDNA specified a receptor that bound LDL normally, but entered the cell slowly. Electron microscopy showed that this receptor was distributed diffusely over the cell surface, whereas the receptor produced by the normal cDNA was concentrated in coated pits. These results support the hypothesis that cytoplasmic domains direct receptors to coated pits, thereby determining the high rate of receptor internalization in animal cells.

Alleles↗
PubMed1986

Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts.

A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts.

Amino Acid Sequence↗
PubMed1986

Molecular cloning of bovine LDL receptor cDNAs.

Using methods described above, a partial cDNA clone for the bovine LDL receptor has been isolated. DNA sequence analysis and Northern blotting experiments are used to confirm the identity of pLDLR-1. Further DNA sequence analysis of pLDLR-1 reveals that the partial cDNA insert encodes 264 amino acids corresponding to the carboxy-terminal 25% of the bovine LDL receptor. Antipeptide antibodies directed against regions of the predicted protein sequence specifically recognize the purified bovine receptor. These findings provide an independent confirmation of the identity of pLDLR-1.

Adrenal Glands↗
PubMed1986

Exon-Alu recombination deletes 5 kilobases from the low density lipoprotein receptor gene, producing a null phenotype in familial hypercholesterolemia.

Among patients with familial hypercholesterolemia, half of the mutant alleles at the low density lipoprotein (LDL) receptor locus produce no immunologically detectable protein. To determine the molecular basis for one such null allele, we have cloned an abnormally short restriction fragment from the genomic DNA of one patient. The DNA sequence revealed a 5-kilobase deletion that joins a coding sequence in exon 13 to an Alu repetitive element in intron 15. The deletion joint is flanked by two inverted repeats that could potentially form a double stem-loop structure that might have predisposed to this deletion. A similar double stem-loop structure can be drawn for a previously described deletion in the LDL receptor gene and for a deletion in the beta-globin gene cluster. We speculate that such double stem-loop structures might contribute to the formation of large deletions in the human genome.

Base Sequence↗
PubMed1986

Deduced primary structure of the alpha subunit of the GTP-binding stimulatory protein of adenylate cyclase.

A bovine adrenal cDNA clone encoding the entire alpha subunit of the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs) was isolated and sequenced. This cDNA directed the synthesis of the larger, 52-kDa form of the polypeptide in COS cells, even though the clone appeared to encode a 46-kDa protein. Comparison of the deduced amino acid sequence of Gs alpha with the alpha subunit of another G protein, transducin, revealed striking homologies.

Adenylyl Cyclases↗
PubMed1986

The effect of social support and the work environment upon burnout among nurses.

Burnout among hospital-based nurses appears to be a serious problem affecting the delivery of health care. Findings from previous empirical research indicate that burnout among these nurses results from reactions to adversities inherent in the hospital work environment, and that burnout can lead nurses to change jobs and/or abandon the practice of nursing. This paper presents and discusses research findings on the effects of various aspects of the hospital work environment on burnout among nurses, and, in addition, evaluates the effects of social support in reducing and/or mitigating the relationship between negative aspects of the work environment and burnout. A multiple regression approach is employed to test the hypothesized model. The data were collected from a sample of nurses (n = 310) employed at Fitzsimons Army Medical Center (FAMC), Aurora, Colorado. The major determinants of burnout were found to be low job enhancement (autonomy, task orientation, clarity, innovation, and physical comfort); work pressure; and lack of supervisor support, along with the interaction term involving the combined effects of job enhancement and supervisor support. These predictors, in conjunction with demographic and job-related variables explained 53% of the variance in emotional exhaustion, a central component of the burnout syndrome.

Burnout, Professional↗
PubMed1986

The LDL receptor in familial hypercholesterolemia: use of human mutations to dissect a membrane protein.

Since the discovery of the LDL receptor 13 years ago, a multidisciplinary approach to its study has revealed much about this important cell-surface protein. Most recently, we have developed tools in the form of full-length cDNAs and cloned genomic DNAs necessary to understand the molecular genetics of this locus. The frequent occurrence of mutations in the LDL receptor gene in patients with FH provides a fertile ground on which to explore the parts of the receptor that are necessary for its function. The analysis of four large deletions has revealed an unexpectedly universal involvement of Alu repeats in their generation. These studies indicate that repetitive DNAs can destabilize a gene through homologous recombination. Inasmuch as the LDL receptor gene is a mosaic of exons shared with at least five other proteins, it is possible that early exon-shuffling events involved recombination between these repetitive elements. Is it possible that the very plasticity that permitted evolution of the LDL receptor also accounts for its frequent disruption by mutation? Further study may help to answer this question. Mutations that disrupt the structure of the protein have been identified. The biochemical and cellular consequences of these mutations reveal crucial aspects of receptor structure. The receptor is clearly divided into quasi-independent domains with discrete functions. Mutations that disrupt the cytoplasmic domain alter the ability of the LDL receptor to cluster in coated pits, but they do not disrupt ligand binding or produce major effects on intracellular transport. Some of the mutations in the external domain disrupt binding but do not affect transport or internalization.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1986

Structure of the Saccharomyces cerevisiae HO gene and analysis of its upstream regulatory region.

The HO gene product of Saccharomyces cerevisiae is a site-specific endonuclease that initiates mating type interconversion. We have determined the nucleotide sequence of a 3,129-base-pair (bp) segment containing HO. The segment contains a single long open reading frame encoding a polypeptide of 586 amino acids, which has unusual (unbiased) codon usage and is preceded by 762 bp of upstream region. The predicted HO protein is basic (16% lysine and arginine) and is calculated to have a secondary structure that is 30% helical. The corresponding transcript is initiated approximately 50 nucleotides prior to the presumed initiation codon. Insertion of an Escherichia coli lacZ gene fragment into the putative HO coding segment inactivated HO and formed a hybrid HO-lacZ gene whose beta-galactosidase activity was regulated by the mating type locus in the same manner as HO (repressed by a 1-alpha 2). Upstream regions of 1,360 and 762 bp conferred strong repression; 436 bp led to partial constitutivity and 301 bp to full constitutivity. Thus, DNA sequences that confer repression of HO by a1-alpha 2 are at least 250 nucleotides upstream of the transcription start point and are within 436 nucleotides of the HO initiation codon. The progressive loss of repression suggests that both the -762 to -436 and the -436 to -301 intervals contain sites for regulation by a1-alpha 2. The HO gene contains two distinct regions that promote autonomous replication of plasmids in S. cerevisiae. These regions contain sequences that are homologous to the two conserved sequences that are associated with ARS activity.

Amino Acid Sequence↗
PubMed1985

The LDL receptor gene: a mosaic of exons shared with different proteins.

The multifunctional nature of coated pit receptors predicts that these proteins will contain multiple domains. To establish the genetic basis for these domains (LDL) receptor. This gene is more than 45 kilobases in length and contains 18 exons, most of which correlate with functional domains previously defined at the protein level. Thirteen of the 18 exons encode protein sequences that are homologous to sequences in other proteins: five of these exons encode a sequence similar to one in the C9 component of complement; three exons encode a sequence similar to a repeat sequence in the precursor for epidermal growth factor (EGF) and in three proteins of the blood clotting system (factor IX, factor X, and protein C); and five other exons encode nonrepeated sequences that are shared only with the EGF precursor. The LDL receptor appears to be a mosaic protein built up of exons shared with different proteins, and it therefore belongs to several supergene families.

Amino Acid Sequence↗
PubMed1985

Cassette of eight exons shared by genes for LDL receptor and EGF precursor.

The amino acid sequences of the human low-density lipoprotein (LDL) receptor and the human precursor for epidermal growth factor (EGF) show 33 percent identity over a stretch of 400 residues. This region of homologous is encoded by eight contiguous exons in each respective gene. Of the nine introns that separate these exons, five are located in identical positions in the two protein sequences. This finding suggests that the homologous region may have resulted from a duplication of an ancestral gene and that the two genes evolved further by recruitment of exons from other genes, which provided the specific functional domains of the LDL receptor and the EGF precursor.

Amino Acid Sequence↗
PubMed1985

Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains.

The molecular size of the plasma LDL (low density lipoprotein) receptor synthesized by cultured fibroblasts from a patient with the internalization-defective form of familial hypercholesterolemia (FH 274) was smaller by 10,000 daltons than the size of the normal LDL receptor. The segment of the gene encoding the truncated portion of the FH 274 receptor was cloned into bacteriophage lambda. Comparison of the nucleotide sequences of the normal and FH 274 genes revealed a 5-kilobase deletion, which eliminated the exons encoding the membrane-spanning region and the carboxyl terminal cytoplasmic domain of the receptor. The deletion appeared to be caused by a novel intrastrand recombination between two repetitive sequences of the Alu family that were oriented in opposite directions. The truncated receptors lack membrane-spanning regions and cytoplasmic domains; they are largely secreted into the culture medium, but a small fraction remains adherent to the cell surface. The surface-adherent receptors bind LDL, but they are unable to cluster in coated pits, thus explaining the internalization-defective phenotype.

Bacteriophage lambda↗
PubMed1985

Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain.

Certain mutant alleles at the low density lipoprotein (LDL) receptor locus produce receptors that bind LDL normally, but fail to cluster in coated pits and therefore cannot transport LDL into cells. We prepared genomic DNA libraries from cells of two individuals with this phenotype (internalization-defective familial hypercholesterolemia) and isolated the segment of the gene encoding the COOH-terminal cytoplasmic domain of the receptor. One mutant gene contains a single base substitution that changes a tryptophan codon (TGG) to a termination codon (TGA). This produces a receptor with only two amino acids in the cytoplasmic domain. The second mutant gene contains a four-base duplication, producing a frameshift that alters the reading frame. The cytoplasmic tail of this receptor has six of the normal amino acids plus eight additional amino acids. These data suggest that the signal for targeting the LDL receptor to coated pits resides in the cytoplasmic domain of the molecule.

Alleles↗
PubMed1985

Polymorphism and evolution of Alu sequences in the human low density lipoprotein receptor gene.

Two clusters of Alu sequences in the human low density lipoprotein (LDL) receptor gene have been analyzed in detail. One Alu cluster is present within the intron separating exons 15 and 16 of the gene and contains a polymorphic Pvu II site. The presence or absence of this site gives rise to two allelic fragments of 14 and 16.5 kilobases, respectively, in genomic Southern blots using cloned cDNA probes. This DNA polymorphic site is caused by a single adenine to guanine transition within an Alu repetitive element. The second cluster of Alu sequences is located in exon 18 of the LDL receptor gene. Southern blotting of primate DNAs suggests that this cluster became associated with the gene about 30 million years ago. Comparison of bovine DNA sequences, which lack this Alu cluster, with those of the human indicates that the Alu sequences inserted in exon 18 in two independent events.

Adult↗
PubMed1985

Human genes involved in cholesterol metabolism: chromosomal mapping of the loci for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase with cDNA probes.

Cellular cholesterol metabolism is regulated primarily through the coordinate expression of two proteins, the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). We have used cDNA probes for the human genes encoding these proteins to determine the precise chromosomal location of the two loci. By in situ hybridization we have regionally mapped the LDL receptor gene, LDLR, to the short arm of chromosome 19 in bands p13.1-p13.3. This result concurs with and extends a previous study in which LDLR was mapped to chromosome 19 by screening somatic cell hybrids with a species-specific monoclonal antibody. We have assigned the HMG-CoA reductase gene, HMGCR, to chromosome 5 by Southern blotting of DNA from a somatic cell hybrid panel and to bands 5q13.3-q14 by in situ hybridizations of the cDNA probe to human metaphase cells with normal and rearranged chromosomes.

Cholesterol↗
PubMed1984

The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA.

The nucleotide sequence of a cloned 5.3 kilobase cDNA for the human low density lipoprotein receptor revealed five domains in the 839 amino acid protein: 322 NH2-terminal amino acids, extremely rich in disulfide-bonded cysteine residues (15%) and including an 8-fold repeat of 40 residues that may contain the LDL binding site; 350 residues homologous to the precursor of mouse epidermal growth factor; a region immediately outside the plasma membrane, rich in serine and threonine and the site of O-linked glycosylation; 22 hydrophobic amino acids, spanning the plasma membrane; and 50 COOH-terminal amino acids, projecting into the cytoplasm. The mRNA for the receptor contains a 3' untranslated region of 2.5 kilobases that includes multiple copies of the Alu family of repetitive DNAs. Transfection of simian COS cells with the human LDL receptor cDNA linked to the SV40 early promoter resulted in expression of functional cell surface receptors.

Amino Acid Sequence↗