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Biomedical subjects

D W Russell

Publications and source records attributed to D W Russell.

At least 145 records · Page 8Linked to original sources
PubMed1988

Overexpression of low density lipoprotein (LDL) receptor eliminates LDL from plasma in transgenic mice.

A complementary DNA encoding the human low density lipoprotein (LDL) receptor under control of the mouse metallothionein-I promoter was injected into fertilized mouse eggs, and a strain of mice expressing high levels of LDL receptors was established. After administration of cadmium, these mice cleared intravenously injected 125I-labeled LDL from blood eight to ten times more rapidly than did normal mice. The plasma concentrations of apoproteins B-100 and E, the two ligands for the LDL receptor, declined by more than 90 percent after cadmium treatment, but the concentration of another apoprotein, A-I, was unaffected. Therefore, overexpression of an endocytotic receptor can dramatically lower the ambient concentration of its ligand in vivo.

Animals↗
PubMed1988

Multiple crm- mutations in familial hypercholesterolemia. Evidence for 13 alleles, including four deletions.

The low density lipoprotein (LDL) receptors in fibroblasts from 132 subjects with the clinical syndrome of homozygous familial hypercholesterolemia were analyzed by immunoprecipitation with an anti-LDL receptor monoclonal antibody. 16 of the 132 cell strains (12%) synthesized no immunodetectable LDL receptor protein, indicating the presence of two mutant genes that failed to produce cross-reacting material (crm- mutations). DNA and mRNA from 15 of the 16 crm- patients, representing 30 crm- genes, were available for further study. Haplotype analysis based on 10 restriction fragment length polymorphisms (RFLPs) suggested that the 30 crm- genes represent 13 mutant alleles. Four of the alleles produced no mRNA. Three of these four mRNA- alleles had large deletions ranging from 6 to 20 kb that eliminated the promoter region of the gene. The fourth mRNA- allele did not contain any deletion or alteration in the promoter sequence; the reason for the mRNA- phenotype was not apparent. Nine alleles were positive for mRNAs, of which three encoded mRNAs of abnormal size. One of the abnormal mRNAs was produced by a gene harboring a deletion, and another was produced by a gene with a complex rearrangement. The third abnormal-sized mRNA (3.1 kb larger than normal) was produced by an allele that had no detectable alterations as judged by Southern blotting. The other six mRNA+ alleles appeared normal by Southern blotting and produced normal-sized mRNA but no receptor protein. The current studies demonstrate that mRNA analysis coupled with haplotype determination by Southern blot analysis can be used to classify crm- mutations at a genetic locus where multiple alleles exist.

Adolescent↗
PubMed1987

Hemimethylation prevents DNA replication in E. coli.

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.

Bacterial Proteins↗
PubMed1987

Deletion in the gene for the low-density-lipoprotein receptor in a majority of French Canadians with familial hypercholesterolemia.

We found a large deletion (more than 10 kilobases) in the gene for the low-density-lipoprotein receptor in 63 percent of French Canadians with heterozygous familial hypercholesterolemia. The deletion also occurred in homozygous form in four of seven French Canadian homozygotes. The deletion removes the promoter and first exon of the gene, and it abolishes the production of messenger RNA for the low-density-lipoprotein receptor. The high frequency of this mutation is presumably related to a founder effect among the 8000 ancestors of present-day French Canadians, who have had relatively little cross-breeding with groups of other national origins. This deletion has not been observed in any other ethnic group. It can be detected by analysis of genomic DNA from blood leukocytes, thus allowing direct diagnosis of familial hypercholesterolemia in a majority of affected French Canadians.

Adult↗
PubMed1987

Three direct repeats and a TATA-like sequence are required for regulated expression of the human low density lipoprotein receptor gene.

The low density lipoprotein receptor is encoded by a "housekeeping" gene that is transcribed in most mammalian cells and is subject to negative feedback regulation by sterols. To determine the basis for this regulated expression, we performed a transfection analysis with hybrid genes containing up to 6500 base pairs of 5' flanking DNA from the low density lipoprotein receptor gene fused to the coding region of the bacterial chloramphenicol acetyltransferase gene. These studies identified a 177-base pair fragment of 5' flanking DNA that is sufficient for expression as well as negative regulation by sterols. The positive elements within this region were further defined by analysis of a series of 15 mutations in which overlapping 10-base pair segments were scrambled by site-specific mutagenesis. These studies identified the positive elements as three imperfect direct repeats of 16 base pairs and a TATA-like sequence. The three repeats contain a sequence that is homologous to the consensus DNA sequence recognized by transcription factor Sp1.

Animals↗
PubMed1987

42 bp element from LDL receptor gene confers end-product repression by sterols when inserted into viral TK promoter.

The LDL receptor, which mediates the cellular uptake of cholesterol, is subject to classic end-product repression when cholesterol accumulates in the cell. We here show that the sensitivity to end-product repression depends upon a 42 bp element in the 5'-flanking region of the human LDL receptor gene. This sequence, designated sterol regulatory element 42 (SRE 42), contains two 16 bp direct repeats that exhibit positive and negative transcriptional activities. Cells transfected with a fusion gene containing SRE 42 inserted into the promoter of the herpes simplex viral TK gene produced abundant mRNA when grown without sterols. When sterols were present, the mRNA was reduced by 57%-95%, depending on the number of copies of SRE in the fusion gene. These transfection data plus DNAase I footprinting experiments suggest a model of end-product repression in which the end product (sterols) opposes the action of a positive transcription factor that binds to a discrete promoter element.

Animals↗
PubMed1987

The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis.

The 50-residue cytoplasmic domain of the low density lipoprotein receptor (amino acids 790-839) directs the receptor to coated pits, thereby facilitating rapid endocytosis of bound low density lipoprotein. To determine the structural features required for this targeting, we produced 24 mutations in the cytoplasmic domain through use of oligonucleotide-directed mutagenesis. The first 22 amino acids of the cytoplasmic domain (residues 790-811) are sufficient for rapid internalization. The amino acid at position 807 is especially critical. Aromatic residues (tyrosine, phenylalanine, or tryptophan) at this position allow rapid internalization. Charged or uncharged aliphatic residues do not substitute. Although the requirements at the neighboring positions (806 and 808) are less stringent, the insertion of proline at position 806 is detrimental. These specificities suggest that the juxtamembranous region of the cytoplasmic domain participates in protein:protein interactions that allow the low density lipoprotein receptor to cluster in coated pits.

Amino Acid Sequence↗
PubMed1987

Duplication of seven exons in LDL receptor gene caused by Alu-Alu recombination in a subject with familial hypercholesterolemia.

A defective LDL receptor gene in a child with familial hypercholesterolemia produces a receptor precursor that is 50,000 daltons larger than normal (apparent Mr 170,000 vs. 120,000). The elongated protein resulted from a 14 kilobase duplication that encompasses exons 2 through 8. The duplication arose from an unequal crossing-over between homologous repetitive elements (Alu sequences) in intron 1 and intron 8. The mutant receptor has 18 contiguous cysteine-rich repeat sequences instead of the normal nine. Seven of these duplicated repeats are derived from the ligand-binding domain, and two repeats are part of the epidermal growth factor precursor homology region. The elongated receptor undergoes normal carbohydrate processing, its apparent molecular weight increases to 210,000, and the receptor reaches the cell surface where it binds reduced amounts of LDL but undergoes efficient internalization and recycling. The current findings support an evolutionary model in which homologous recombination between repetitive elements in introns leads to exon duplication during evolution of proteins.

Base Sequence↗
PubMed1987

Alu-Alu recombination deletes splice acceptor sites and produces secreted low density lipoprotein receptor in a subject with familial hypercholesterolemia.

A Japanese subject with homozygous familial hypercholesterolemia was found to have a 7.8-kilobase deletion in the gene for the low density lipoprotein receptor. The deletion joins intron 15 to the middle of exon 18, which encodes the 3' untranslated region, thereby removing all 3' splice acceptor sites distal to intron 15. By S1 nuclease mapping, we demonstrated that the 5' splice donor site of intron 15 is no longer used. Instead a continuous transcript is produced in which exon 15 is followed by the remaining segments of intron 15 and exon 18. The translational reading frame of exon 15 continues for 165 nucleotides into intron 15 before a termination codon is reached. This mRNA should produce a truncated receptor that lacks the normal membrane-spanning region and cytoplasmic domain and that has 55 novel amino acids at its COOH terminus. A cDNA expression vector containing this sequence produced a receptor that behaved similarly to the truncated protein produced by the Japanese patient, i.e. greater than 90% of the receptor was secreted from the cell, and the receptors remaining on the surface showed defective internalization. The deletion in this subject resulted from a recombination between two repetitive sequences of the Alu family, one in intron 15 and the other in exon 18. To date, Alu sequences have been observed at the deletion joints of all four gross deletions in the low density lipoprotein receptor gene that have been characterized. Within these Alu sequences, six out of the seven breakpoints have occurred in the left arm. These data suggest that recombination between Alu sequences may be a frequent cause of deletions in the human genome.

Amino Acid Sequence↗
PubMed1987

The Lebanese allele at the low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum.

We here describe a mutant low density lipoprotein receptor gene that produces a shortened receptor protein lacking three domains: the region of clustered O-linked carbohydrates, the membrane-spanning region, and the cytoplasmic tail. The defect is attributable to a single nucleotide substitution that creates a premature termination codon at amino acid 660, eliminating 180 residues from the mature protein. The truncated protein retains only two domains: a complete ligand-binding region (residues 1-292) and a partial epidermal growth factor precursor homology region (residues 293-659). The termination codon occurs in the middle of a cysteine-rich sequence that is part of the epidermal growth factor precursor homology domain. The mutant protein is present in markedly reduced amounts and may be translated at a reduced rate. After synthesis, most of the receptor remains within the cell for several hours with its N-linked carbohydrate in an unprocessed endoglycosidase H-sensitive form. This finding suggests that the shortened receptor leaves the endoplasmic reticulum at an abnormally slow rate, which is likely attributable to abnormal folding of the truncated protein. The mutation creates a new restriction site for the enzyme HinfI, thus permitting diagnosis by Southern blotting of genomic DNA. Two copies of this mutant gene were present in each of four unrelated Arab patients with homozygous familial hypercholesterolemia (three from Lebanon and one from Syria). We believe that this mutation, hereafter referred to as the "Lebanese allele," is responsible for the extraordinarily high incidence of familial hypercholesterolemia in Lebanon.

Base Sequence↗
PubMed1987

Cloning and expression of recombinant, functional ricin B chain.

The cDNA encoding the B chain of the plant toxin ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with [35S]methionine and [35S]-cysteine and demonstrating the secretion of a protein with a Mr of 30,000-32,000 that was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B-chain antibody and the amount of recombinant B chain secreted by the COS-M6 cells was determined by a radioimmunoassay. Virtually all of the recombinant B chain formed active ricin when mixed with native A chain; it could also bind to the galactose-containing glycoprotein asialofetuin as effectively as native B chain. These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function.

Animals↗
PubMed1987

mRNA for low density lipoprotein receptor in brain and spinal cord of immature and mature rabbits.

Hybridization studies with [32P]cDNA probes revealed detectable amounts of mRNA for the low density lipoprotein (LDL) receptor in the central nervous system (CNS) of rabbits. mRNA levels were highest in the medulla/pons and spinal cord, which were the most heavily myelinated regions that were studied. Lower, but detectable levels were present in cerebral cortex, hypothalamus, thalamus, midbrain, and cerebellum. In the medulla/pons and spinal cord, the levels of receptor mRNA were in a range comparable to that detected in the liver. The levels of receptor mRNA in whole brain were constant from 3 days of age to adulthood and, thus, did not vary in proportion to the rate of myelin synthesis. LDL receptor mRNA in the CNS was produced by the same gene that produced the liver and adrenal mRNA as revealed by the demonstration of a deletion in the neural mRNA of Watanabe-heritable hyperlipidemic (WHHL) rabbits identical to the deletion in the LDL receptor gene of these mutant animals. Using antibodies directed against the bovine LDL receptor, we showed that LDL receptor protein is present in the medulla/pons of adult cows. The cell types that express LDL receptors in the CNS and the functions of these receptors are unknown.

Animals↗
PubMed1986

Deletion of exon encoding cysteine-rich repeat of low density lipoprotein receptor alters its binding specificity in a subject with familial hypercholesterolemia.

The proposed ligand binding domain of the low density lipoprotein (LDL) receptor consists of a 40-amino acid cysteine-rich unit that is repeated with some variation seven times. We describe here a mutant allele at the LDL receptor locus in which one of the seven repeats has been deleted. This mutation was found in a patient with the clinical syndrome of homozygous familial hypercholesterolemia. By molecular cloning, we show that the deletion arose by homologous recombination between repetitive Alu sequences in intron 4 and intron 5 of the gene. The deletion removes exon 5, which normally encodes the sixth repeat of the ligand binding domain. In the resultant mRNA, exon 4 is spliced to exon 6, preserving the reading frame. This mRNA produces a shortened protein that reaches the cell surface and reacts with anti-receptor antibodies but does not bind LDL, which contains apoprotein B-100 as its major protein component. Surprisingly, the deleted protein retains the ability to bind and internalize beta-migrating very low density lipoprotein, a lipoprotein that contains apoprotein E as well as apoprotein B-100. These data support the hypothesis that the seven repeated sequences in the receptor constitute the LDL binding domain. The data further indicate that the sixth repeat is required for binding of LDL, but not beta-migrating very low density lipoprotein, and that deletion of a single cysteine-rich repeat can alter the binding specificity of the LDL receptor.

Alleles↗