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O Smithies

Publications and source records attributed to O Smithies.

At least 127 records · Page 7Linked to original sources
PubMed1990

Molecular analysis of deletions in the human beta-globin gene cluster: deletion junctions and locations of breakpoints.

DNA fragments that contain the deletion junction regions of four independent deletions involving the human beta-globin gene cluster have been isolated and cloned. The fragments were isolated from individuals with the conditions referred to as Sicilian (delta beta)zero-thalassemia, Turkish G gamma+(A gamma delta beta)zero-thalassemia, Black G gamma+(A gamma delta beta)zero-thalassemia, and HPFH-2. The sequences of the deletion junctions and of the normal DNA surrounding their 3' breakpoints were determined and compared to the previously determined sequences of normal DNA surrounding their 5' breakpoints. These comparisons show that the deletions were the result of nonhomologous recombinational events. Two of the deletion junctions contain "orphan" nucleotides, while the other two show very limited amounts of "junctional homology." Both types of junctions are common among recombination events in mammalian cells and we discuss a simple joining scheme that could account for the junctions reported here. Unlike other deletions in this cluster and in other gene clusters, none of the eight deletion breakpoints examined here occurred within Alu family repeats. To examine the significance of deletion breakpoints within various sequence categories, we analyzed the data from a well-defined set of deletions within this locus. In contrast to deletions in the alpha-globin gene cluster, the occurrence of breakpoints in Alu family repetitive sequences is not statistically significant within the beta-globin gene cluster. However, breakpoints do occur within transcriptional units of the beta-globin gene cluster more frequently than expected by chance alone. We conclude from our analysis that the mechanisms of DNA joining are not locus or location specific, but at least a portion of the mechanisms of chromosomal breakages do show locus specificity.

Base Sequence↗
PubMed1990

A physical map of the human salivary proline-rich protein gene cluster covers over 700 kbp of DNA.

By using a linking library, we have experimentally linked, ordered, and spaced four of the six loci that constitute the human salivary proline-rich protein (PRP) multigene family. The methods used for mapping these four PRP genes may be useful in other multigene systems in which no probes unique to each member of genes are available, but in which some enzyme site that occurs only once in each member of the family can be found. The remaining two PRP loci have been provisionally mapped and linked within the gene cluster primarily on the basis of the resulting order giving a simple map. The order of the six loci that most simply accounts for our data is PRB2, PRB1, PRB4, PRH2, PRB3, and PRH1. The PRP gene cluster spans at least 700 kbp on chromosome 12 at p13.2. A scheme for the evolution of the cluster that requires an initial gene duplication followed by three unequal but homologous crossovers is given.

Blotting, Southern↗
PubMed1990

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.

Animals↗
PubMed1989

Replication of plasmid DNA in fertilized Xenopus eggs is sensitive to both the topology and size of the injected template.

The behavior of various plasmid templates was examined following their microinjection into fertilized eggs of the frog Xenopus laevis using an assay that permits the examination of both replicated and unreplicated plasmids in single eggs. Our results show that both the size and the topology of the template drastically affect the fate of the injected plasmid. Only a small proportion of injected monomeric supercoiled plasmids underwent replication during 6 h of incubation, although not all injected cells supported replication. Nicked circles were less stable than supercoiled molecules, and we could not detect their replication. Linear monomeric molecules polymerized into large, randomly oriented multimers which were extensively, but not entirely, replicated. Similar results were obtained when linear templates were ligated into polymeric forms in vitro prior to injection. Thus large molecules or molecules which, due to their topology, could be converted into high molecular weight forms following injection were preferred templates for replication. On rare occasions tandemly repeated, high molecular weight DNA was generated following the injection of supercoiled plasmid monomers. This large DNA was shown to be almost entirely replicated.

Animals↗
PubMed1989

Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells.

Embryonic stem cells (derived from 129/Ola mice) containing a mutant hypoxanthine phosphoribosyltransferase gene that had been corrected in vitro in a planned manner by homologous recombination were injected into blastocysts obtained from C57BL/6J mice. The injected blastocysts were introduced into pseudopregnant female mice to complete their development. Eleven surviving pups were obtained. Nine were chimeras: six males and three females. Two of the males transmitted the embryonic stem cell genome containing the alteration in the hypoxanthine phosphoribosyltransferase gene to their offspring at high frequencies. These experiments demonstrate that a preplanned alteration in a chosen gene can be made in the germ line of an experimental animal by homologous recombination in an embryonic stem cell.

Animals↗
PubMed1989

Inactivating the beta 2-microglobulin locus in mouse embryonic stem cells by homologous recombination.

We have inactivated, by gene targeting, the endogenous beta 2-microglobulin gene in a mouse embryonic stem cell line. A cloned fragment of the beta 2-microglobulin gene with the coding sequence disrupted by the insertion of the neomycin-resistance gene was used to transfect the embryonic stem cells. G418-resistant colonies were selected and then screened using the polymerase chain reaction to identify those in which the incoming DNA had integrated into the embryonic stem cell genome by homologous recombination. Of a total of 234 G418-resistant colonies screened, 2 correctly targeted colonies were identified. Chimeric mice carrying the inactivated beta 2-microglobulin gene have been obtained from both of these targeted embryonic cell lines. Breeding of offspring from such animals will allow investigation of the effects of homozygous loss of beta 2-microglobulin.

Animals↗
PubMed1988

Molecular genetics of the apolipoprotein B gene in pigs in relation to atherosclerosis.

Immunologically defined alleles of the pig apolipoprotein B (ApoB) locus (apoB) are correlated with different blood cholesterol levels and predisposition towards premature coronary heart disease. We show here that these alleles are associated with differences in the apoB gene by identifying six restriction fragment length polymorphisms at the pig apoB locus. We have sequenced a 2.4-kb fragment encompassing exons 11 through 14 of one allele, and 7.1 kb from the 3' one-third of exon 26 to about 1 kb past the last exon from another allele. The decoded amino acids of the pig and human ApoB proteins are identical at 70% of these positions. One region close to the C-terminus of the protein is surprisingly different in pigs and humans (57% identity) but the C-terminal region is relatively well conserved (74% identity). Neither of the two putative low-density lipoprotein (LDL) receptor-binding sites is completely conserved in pigs and humans, but identical stretches of amino acids occur near these sites more frequently than in the other sequenced regions. We compare the nucleotide sequences of the region encompassing the putative LDL receptor-binding sites from four pig alleles, including one implicated directly in atherosclerosis. None of the differences appears to account for the hypercholesterolemic phenotype. We conclude that significant differences in the physiology of LDL particles result from changes outside the putative receptor-binding region.

Alleles↗
PubMed1988

Recombinant fragment assay for gene targetting based on the polymerase chain reaction.

The modification of chromosomal genes by homologous recombination between exogenous DNA and a target locus provides a powerful approach to the study of gene function. One of the current limitations of this gene targetting is the difficulty of identifying cells containing the correctly modified target locus when the modified gene is not amenable to either direct or indirect selection. We here describe a procedure for identifying correctly modified cells that depends on amplifying by the polymerase chain reaction (PCR) predictable fragments of DNA present only in the desired recombinants. This recombinant fragment assay can detect targetted modification using only a few cells, either alone or mixed with tens of thousands of unmodified cells; it does not depend on the phenotype of the modified gene, or on its expression in the target cells. The PCR amplification needed for the procedure is carried out with a heat stable polymerase and a simple automatic temperature-shift apparatus which is described.

Animals↗
PubMed1988

Regulated expression of genes inserted at the human chromosomal beta-globin locus by homologous recombination.

We have examined the effect of the site of integration on the expression of cloned genes introduced into cultured erythroid cells. Smithies et al. [Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. & Kucherlapati, R.S. (1985) Nature (London) 317, 230-234] reported the targeted integration of DNA into the human beta-globin locus on chromosome 11 in a mouse erythroleukemia-human cell hybrid. These hybrid cells can undergo erythroid differentiation leading to greatly increased mouse and human beta-globin synthesis. By transfection of these hybrid cells with a plasmid carrying a modified human beta-globin gene and a foreign gene composed of the coding sequence of the bacterial neomycin-resistance gene linked to simian virus 40 transcription signals (SVneo), cells were obtained in which the two genes are integrated at the beta-globin locus on human chromosome 11 or at random sites. When we examined the response of the integrated genes to cell differentiation, we found that the genes inserted at the beta-globin locus were induced during differentiation, whereas randomly positioned copies were not induced. Even the foreign SVneo gene was inducible when it had been integrated at the beta-globin locus. The results show that genes introduced at the beta-globin locus acquire some of the regulatory properties of globin genes during erythroid differentiation.

Animals↗
PubMed1988

Targeted mutation of the Hprt gene in mouse embryonic stem cells.

The hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene has been mutated in mouse blastocyst-derived embryonic stem cells by site-directed homologous recombination. Embryonic stem cells were electroporated in the presence of a targeting DNA fragment containing two specific features: (i) The targeting DNA contained a promoterless neomycin phosphotransferase (neo) gene that, when located within the endogenous Hprt locus, could be transcribed from the promoter of the target locus. (ii) The targeting fragment had two short regions of homology with the endogenous Hprt gene: one, 132 base pairs long and the other, 1.2 kilobase pairs long. Targeted cells in which the designed homologous recombination event occurred were isolated either by selection with G418 followed by 6-thioguanine or by selection with 6-thioguanine alone. Even though less than 2 kilobases of homology existed between the exogenous and target DNAs, an average of 2.6 embryonic stem cells were successfully targeted for every 10(5) colonies surviving electroporation. Six of the Hprt- cell lines showed homologous recombination. These six lines were further analyzed by nucleotide sequencing a fragment that spans one crossover point after amplification by the polymerase chain reaction. Four lines had the expected sequence, whereas two lines had small deletions abutting the 132-base-pair region of homology.

Amino Acid Sequence↗
PubMed1988

Many protein products from a few loci: assignment of human salivary proline-rich proteins to specific loci.

Earlier studies of protein polymorphisms led to the description of 13 linked loci thought to encode the human salivary proline-rich proteins (PRPs). However, more recent studies at the DNA level have shown that there are only six genes which encode PRPs. The present study was undertaken in order to reconcile these observations. Nucleotide and decoded amino acid sequences from each of the six genes were compared with the available protein sequence data for PRPs. This analysis allowed assignment of the PmF, PmS and Pe proteins to the PRB1 locus, the G1 protein to the PRB3 locus, the Po protein to the PRB4 locus, the Ps protein to the PRB2 locus, and the CON1 and CON2 proteins to the PRB4 locus. Correlations between insertion/deletion RFLPs and PRP protein phenotypes were observed for the PmF, PmS, Gl and CON2 proteins. Our overall analysis indicates that in many instances several proteins previously considered to be the products of separate loci are actually proteolytic cleavage products of a large precursor specified by one or other of the six genes identified at the DNA level. Our analysis also demonstrates that some of the "null" alleles proposed to occur at 11 of the 13 loci in the earlier genetic studies, are actually productive alleles having alterations at proteolytic cleavage sites within the relevant precursor protein. The absence of cleavage leads to the persistence of longer precursor peptides not resolved electrophoretically, concurrently with an absence of the smaller PRPs seen when cleavage occurs.

Alleles↗
PubMed1988

Length polymorphisms in human proline-rich protein genes generated by intragenic unequal crossing over.

Southern blot hybridization analysis of genomic DNAs from 44 unrelated individuals revealed extensive insertion/deletion polymorphisms within the BstNI-type loci (PRB1, PRB2, PRB3 and PRB4) of the human proline-rich protein (PRP) multigene family. Ten length variants were cloned, including alleles at each of the four PRB loci, and in every case the region of length difference was localized to the tandemly repetitious third exon. DNA sequences covering the region of length variation were determined for seven of the alleles. The data indicate (1) that the PRB loci can be divided into two subtypes, PRB1 plus PRB2, and PRB3 plus PRB4, and (2) that the length differences result from different numbers of tandem repeats in the third exons. Variant chromosomes were also identified with different numbers of PRP loci resulting from homologous but unequal exchange between the PRB1 and PRB2 loci. The overall data are compatible with the observed length variants having been generated via homologous but unequal intragenic exchange. The results also indicate that these crossover events are sensitive to the amount of homology shared between the interacting DNA strands. Allelic length variants have arisen independently at least 20 times at the PRB loci, but only one has been detected at a PRH locus. Comparison of the detailed structures of the repetitious regions in PRB and PRH loci shows that the repeats in PRB genes are very similar to each other in sequence and in length. The PRH genes contain fewer repeats, which differ considerably in their individual lengths. These differences suggest that the larger number of length variants in PRB genes is related to their greater ease of homologous but unequal pairing compared to PRH genes.

Alleles↗
PubMed1988

Cystatin superfamily. Evidence that family II cystatin genes are evolutionarily related to family III cystatin genes.

Human saliva contains at least three molecular species of cystatin S-type cysteine proteinase inhibitor (cystatin S, cystatin SN and cystatin SA), which have similar but distinct amino-acid sequences. The nucleotide sequences of the CST 1 gene for cystatin SN and the CST 2 gene for cystatin SA are highly homologous to each other and to the corresponding regions of the cDNA for cystatin C and the EcoRI-PstI fragment from the cystatin C gene. Three cystatin-like domains in the kininogen gene and the salivary cystatin genes share the same gene organizations. These data demonstrate that family II cystatin genes are evolutionarily related to family III cystatin genes.

Amino Acid Sequence↗
PubMed1987

Human cysteine-proteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family.

Three genes from the human cystatin gene family of cysteine-proteinase inhibitors have been isolated from a bacteriophage lambda library containing HindIII digests of human genomic DNA. Two of the genes code for salivary cystatin SN and SA, the third is a pseudogene. The cloned genes were identified with a probe made from a salivary cystatin cDNA. The complete nucleotide sequence of the gene that codes for the precursor form of the neutral salivary protein, cystatin SN, was determined. The gene, which we name CST1, contains three exons and two intervening sequences. The expected CAT and ATA boxes are present in the 5'-flanking region of the gene. Partial nucleotide sequence determination of a second gene revealed that it codes for the precursor form of the acidic salivary protein, cystatin SA. This gene, which we name CST2, has the same gene organization as CST1. The complete nucleotide sequence of a third gene was determined. It does not contain a typical ATA box, and in addition, a premature stop codon and a frameshift deletion mutation occur within the gene. These inactivation mutations show that this gene, which we name CSTP1, is a cystatin pseudogene. These data combined with our genomic Southern-blot analyses show that the cystatin genes form a multigene family with at least seven members.

Base Sequence↗
PubMed1987

Accurate modification of a chromosomal plasmid by homologous recombination in human cells.

We have examined the consequences of modifying mammalian cellular DNA sequences by homologous recombination. A plasmid carrying a 248-base-pair deletion in the neomycin phosphotransferase (neo) gene was introduced into hamster and human cells. The integrated, defective neo gene was used as a target for modification by a second round of transfection with a plasmid carrying a different (283-base-pair) deletion in the neo gene. Recombinants resulting in an intact neo gene were selected by their G418 resistance phenotype. The best ratio of homologous to nonhomologous recombination events was about 1:80. Analyses of the functional neo genes in various independent cell lines establish that simple crossovers (single and double) generated the wild-type neo genes.

Animals↗
PubMed1986

Gene conversions and their relation to homologous chromosome pairing.

Gene conversion is the non-reciprocal transfer of DNA sequences from one gene to a related gene elsewhere in the genome. Molecular evidence for its occurrence in higher eukaryotes was first described by our laboratory in 1980 in the two linked human foetal gamma-globin genes. Over a kilobase of DNA was converted in this initial example. Other investigators have since described more examples of gene conversion including some in which the sequence that was transferred is much shorter. We have now accumulated evidence for a series of such small gene conversions in the human foetal globin gene pair. The number of small gene conversions that we have been able to detect leads us to suggest that gene conversions are the consequence of a general mechanism whereby DNA strand invasions enable chromosomes to find their homologues during meiosis.

Biological Evolution↗
PubMed1986

Direct alteration of a gene in the human genome.

Direct alteration of a gene in the human genome requires an understanding of the role of the gene in metabolism. A gene may need to be introduced into a specific tissue or alternatively it may be possible to use accessible tissue such as bone marrow. The level of gene expression required also needs to be known as does the position in the genome into which the gene is to be inserted. Insertion of DNA needs to be of high efficiency and accuracy. Various methods are available including virus, the use of inert adjuvant, microinjection and electroporation. The procedure with the most potential for accuracy is the use of specially designed plasmids. The example of the use of such a plasmid in achieving target modification of the beta-globin gene is given. The method has high accuracy but low efficiency.

DNA↗