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Biomedical subjects

O Smithies

Publications and source records attributed to O Smithies.

At least 163 records · Page 9Linked to original sources
PubMed1982

Human globin psi B2 is not a globin-related sequence.

We have determined the complete nucleotide sequence of 3.4 kilobase pairs of DNA covering the region of the human beta globin gene cluster where a human globin-related sequence psi beta 2 was thought to lie (Fritsch, Lawn, and Maniatis (1980) Cell 19, 959-972). Analysis of the resulting data reveals no evidence for any globin-related sequences in this region. The region does, however, contain several stretches of poly (dA-dT). We have confirmed the observations of Fritsch et al. that DNA from the psi beta 2 region hybridizes to the poly (dA-dT)-tailed human fetal globin cDNA plasmid, pJW151 (Wilson et al., (1978) Nucl. Acids Res. 5, 563-581) under conditions of low stringency, but we find that this hybridization is abolished by the addition of poly(rA). We conclude that psi beta 2 is not a globin pseudogene, and that the earlier investigators were probably misled by hybridization between the poly (dA-dT) stretches within the psi beta 2 region and the tails used in constructing the cDNA plasmid.

Base Sequence↗
PubMed1982

The control of globin and other eukaryotic genes.

The hypothesis is advanced that eukaryotes control different parts of their genomes by the selective use of origins of replication. Replication of a gene from an origin upstream of the gene is postulated to open it for transcription under appropriate circumstances; replication of a gene from an origin downstream of the gene closes it. The applicability of this hypothesis to the control of the human beta-globin cluster of genes is considered.

Animals↗
PubMed1982

Introduction and expression of a fetal human globin gene in mouse fibroblasts.

An 8.5-kilobase segment of cloned human DNA including the complete G gamma-globin gene was introduced into LMTK- cells by the calcium phosphate precipitation method in the presence or absence of carrier DNA. Transfectants containing one or more copies of intact G gamma-globin genes were obtained either by ligation of the human DNA segment to a plasmid containing the herpes simplex virus thymidine kinase gene or by nonligated cotransfer. The integrity of the integrated gamma-globin gene was established by Southern blotting experiments. Expression of the herpes simplex virus thymidine kinase and human gamma-globin genes was evaluated by Northern blotting and solution hybridization. Of 23 transfectants analyzed, 21 produced a 9S gamma-globin RNA migrating like authentic gamma-globin mRNA on denaturing agarose gels. The gamma-globin RNA is polyadenylated and present in the cytoplasm of the transfected cells; it accumulates to a level 10 times that of thymidine kinase mRNA, or about 5 to 50 molecules per transfected cell. By using plasmids in which the gamma-gene is inserted in either transcriptional orientation with respect to the thymidine kinase gene, it was possible to show that transcription occurred from the gamma-gene promoter.

Animals↗
PubMed1981

Base substitutions, length differences and DNA strand asymmetries in the human G gamma and A gamma fetal globin gene region.

We have studied differences arising subsequent to the 5 kilobase pair (kb) duplication that led to the human G gamma and A gamma fetal globin genes. The local occurrence of base substitutions in the duplicated 5 kb region correlates positively with the local AT base pair content. This correlation also occurs in two mouse beta-globin genes and in two mouse immunoglobulin genes. The relationship is valid for transcribed or nontranscribed DNA and for DNA that contains only coding sequences. Length differences in the fetal globin duplicated regions correlate positively with the occurrence of short direct repeats of greater than or equal 5 base pairs. Path analysis of the interrelationships of base composition, base substitutions, repeats and length differences provides an integrated view of the relative effects on chromosomal changes of these variables and of selection. The distributions along the chromosome of simple sequences and of base compositions show highly significant local asymmetries between the transcribed and nontranscribed strands of the DNA, which permit us to divide the fetal globin gene region into chromosomal domains. Comparable domains are present in DNA from other sources, including the mammalian viruses SV40 and polyoma virus strain A-2 in which some of the domains appear related to discrete functions.

Animals↗
PubMed1981

A history of the human fetal globin gene duplication.

The nucleotide sequence of 11.4 kilobase pairs (kb) of human DNA that includes the two fetal globin genes, G gamma and A gamma, shows that they are part of a 5 kb tandem duplication. A small segment of DNA occurs three times, on either side of and between the two duplicates. We present two models that account for these observations. One model is simple but requires the assumption of a preexisting repetitive element; the other is more complex but does not require the assumption of preexisting repeats. Over much of the 5 kb duplicated region, the present duplicate copies differ by an average of 14% of their bases, from which we calculate that the duplication was first formed about 34 million years ago. However, 1.5 kb of DNA are vitually identical in the two genes analyzed here, probably as a consequence of an intergenic exchange (gene conversion) that replaced part of the diverging A gamma gene with the corresponding part of the G gamma gene. This conversion took place around 1 million years ago. A sequence of repeated dinucleotides may be one signal involved in exchanges leading to this and similar conversions. Cycles of duplication, triplication, deletion and gene conversion on probably common in many multigene families.

Base Sequence↗
PubMed1980

A mouse alpha-globin-related pseudogene lacking intervening sequences.

A mouse alpha-globin-related pseudogene (psi alpha 30.5) completely lacks intervening sequences, and could not code for a functional globin poypeptide because of frameshifts. The widespread occurrence of globin pseudogenes in other species suggests that they are not 'dead' genes but may be important in controlling globin expression.

Animals↗
PubMed1980

Human fetal G gamma- and A gamma-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes.

We present the nucleotide sequences of the G gamma-and A gamma-globin genes from one chromosome (A) and of most of the A gamma gene from the other chromosome (B) of the same individual. All three genes have a small, highly conserved intervening sequence (IVS1) of 122 bp located between codons 30 and 31 and a large intervening sequence (IVS2) of variable length (866-904 bp) between codons 104 and 105. A stretch of simple sequence DNA occurs in IVS2 which appears to be a hot spot for recombination. On the 5' side of this simple sequence, the allelic A gamma genes differ considerably in IVS2 whereas the nonallelic G gamma and A gamma genes from chromosome A differ only slightly. Yet on the 3' side of the simple sequence, the allelic genes differ only slightly whereas the nonallelic genes differ considerably. We hypothesize that the 5' two thirds of the A gamma gene on chromosome A has been "converted" by an intergenic exchange to become more like the G gamma gene on its own chromosome A than it is like the allelic A gamma gene on the other chromosome B. Our sequence data suggest that intergenic conversions occur in the germ line. The DNA sequence differences between two chromosomes from a single individual strongly suggest that DNA sequence polymorphisms for localized deletions, additions and base substitutions are very common in human populations.

Alleles↗
PubMed1980

The structure and evolution of the human beta-globin gene family.

We present the results of a detailed comparison of the primary structure of human beta-like globin genes and their flanking sequences. Among the sequences located 5' to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 +/- 1 and 77 +/- 10 bp, respectively, 5' to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calculation of the divergence times of individual beta-like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence comparisons. In addition, the evolutionary relationship of the embryonic epsilon-globin gene to the other human beta-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of beta-like globin genes during evolution.

Animals↗
PubMed1978

Cloning human fetal gamma globin and mouse alpha-type globin DNA: preparation and screening of shotgun collections.

Shotgun collections of Charon 3A bacteriophages containing Eco RI fragments of human and mouse DNA were constructed with the use of in vitro packaging. Plaques were screened by hybridization, and globin-specific clones were isolated from both human (Charon 3AHs51.1) and mouse (Charon 3AMm30.5). The fragments cloned were detected in unfractionated genomic DNA by the Southern method of hybridization.

Animals↗
PubMed1978

Cloning human fetal gamma globin and mouse alpha-type globin DNA: characterization and partial sequencing.

Two globin-related clones isolated from collections of bacteriophages containing unfractionated Eco RI fragments of human and mouse DNA were characterized. Charon3AHs51.1Hbgamma includes 2.7 kilobase pairs of human DNA containing a large part of a fetal gamma globin chain structural gene; Charon 3AMm30.5 includes 4.7 kilobase pairs of mouse DNA related to alpha globin. The human fetal gamma globin gene has within its coding region two intervening sequences of noncoding DNA, IVS 1 and IVS 2, of approximately 1-0 and 900 base pairs. Sequence IVS 1 is located at the position of one of the two intervening sequences occurring in adult globin genes; IVS 2 is located at the position of the other.

Amino Acid Sequence↗
PubMed1977

Nonrandom distribution of chromosomal proteins during cell replication.

The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has been examined using the method described by Taichman and Freedlender (Taichman, L., and Freedlender, E.F. (1976), Biochemistry 15, 447). Cells are grown for several generations in [14C]lysine and thymidine, and then for one generation in the presence of [3H]lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in cold amino acid and BrUdRib. This protocol produces equal amounts of unifilarly (heavy-light) and bifilarly (heavy-heavy) substituted DNA. Chromatin containing the two types of DNA are separated by sucrose-gradient centrifugation after ultraviolet irradiation. The results indicate that some of the chromatin proteins can segregate with the DNA strand synthesized in the same generation when the cells subsequently replicate. Using chromatin with a protein to DNA ratio of 2.6, in different experiments, 5-22% of the chromatin proteins were estimated to segregate with the appropriate DNA strand, while the remaining proteins were randomly distributed to daughter chromatin. The segregating proteins have not been specifically identified but they migrate in sodium dodecyl sulfate gel electrophoresis in the region where the four smaller histones migrate.

Animals↗
PubMed1977

Charon phages: safer derivatives of bacteriophage lambda for DNA cloning.

The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.

Chromosome Mapping↗
PubMed1976

beta2-microglobulin locus on human chromosome 15.

We have developed an autoradiographic/electrophoretic assay capable of distinguishing mouse and human beta2-microglobulin (beta2m) in spent culture media. The method is applicable to mouse and human lines and to hybrid cell lines made from them. With this technique, mouse/human hybrid cell lines were tested for the presence of human beta2m. Isozyme and karyotype analyses were also carried out with the hybrids. The combined results of these studies show that the structural gene for human beta2m is on the long arm of chromosome 15.

Beta-Globulins↗