Search PubMed⌕ Search

Biomedical subjects

T Maniatis

Publications and source records attributed to T Maniatis.

At least 181 records · Page 10Linked to original sources
PubMed1982

The structure of the human zeta-globin gene and a closely linked, nearly identical pseudogene.

DNA sequencing studies indicate that only one of two closely linked human embryonic alpha-like globin genes, zeta (zeta), encodes a functional polypeptide. The other is a pseudogene (psi zeta) that differs by only 3 bp in the protein coding sequence, one of which converts the codon for amino acid 6 into a chain termination codon. Both zeta-globin genes differ from all other alpha-like genes thus far reported in that they contain large introns consisting, in part, of simple repeat sequences. Intron 1 of each gene contains a variation of the repeat sequence ACAGTGGGGAGGGG, while intron 2 contains the repeat sequence CGGGG. Comparison of the human zeta- and alpha-globin gene sequences reveals that the embryonic and adult alpha-like genes began to diverge from each other relatively early in vertebrate evolution (400 million years ago). In contrast, the beta-like embryonic globin gene, epsilon (epsilon), is the product of a much more recent evolutionary event (200 million years ago). Thus, even though the temporal and quantitative expression of zeta- and epsilon-globin genes must be coordinately controlled during development, their evolutionary histories are clearly distinct.

Amino Acid Sequence↗
PubMed1982

A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.

We have studied the structure and transcription of a cloned human beta-globin gene from a fetus diagnosed for beta 0 thalassemia. The sequence of the beta 0 gene differs from that of a normal beta-globin gene at positions 1 and 74 of the second intervening sequence (IVS2). The position 1 change alters the GT dinucleotide conserved at 5' splice sites, while the position 74 change is a common sequence polymorphism. When the cloned beta 0 gene is introduced into HeLa cells by use of an SV40-derived plasmid vector, two abnormally spliced cytoplasmic beta-globin RNAs are detected. The predominant RNA differs from normal beta-globin mRNA by the insertion of the first 47 nucleotides of IVS2 between exons 2 and 3. The less abundant RNA comprises the normal first exon spliced directly to the third. Analysis of nuclear RNA suggests that the beta 0 transcript is inefficiently spliced and that the removal of the two intervening sequences is coupled.

Base Sequence↗
PubMed1982

Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells.

We report the construction of a bovine papillomavirus (BPV)-derived recombinant plasmid that propagates as an extrachromosomal element in both mouse and bacterial cells. Plasmids composed of a subgenomic transforming fragment of BPV DNA, a deletion derivative of pBR322, and a 7.6-kilobase fragment of DNA from the human beta-globin gene cluster efficiently induce focus formation on mouse C127 cells. BPV-beta-globin hybrids are maintained in the transformed cells as plasmids with a copy number of about 10-30 per cell. Plasmids indistinguishable from the input DNA have been recovered by transformation of bacteria with low molecular weight DNA from transformed mouse cells. The human beta-globin gene linked to BPV DNA is transcribed from its own promoter at a high level in these cells. The expression of BPV-linked cellular genes in conjunction with the ability to shuttle DNA between bacteria and mammalian cells may provide a rapid means of analyzing and recovering genes that confer an identifiable phenotype upon mammalian cells.

Animals↗
PubMed1982

Regulated expression of an extrachromosomal human beta-interferon gene in mouse cells.

Beta(fibroblast)-interferon mRNA and protein are induced by the synthetic double-stranded RNA poly(I) X poly(C) in cultured human fibroblasts. To study the mechanism of this induction, we have isolated a human beta-interferon gene and inserted it in a vector plasmid containing DNA of the bovine papilloma virus. After removal of bacterial plasmid sequences, the bovine papilloma virus-beta-interferon recombinant was used to morphologically transform mouse fibroblasts. Analysis of DNA from the transformed cell lines indicated that this recombinant is propagated as a stable multicopy extrachromosomal element. Human beta-interferon mRNA and protein are inducible by poly(I) X poly(C) in all of these cell lines, and the mRNA is indistinguishable from beta-interferon mRNA synthesized by induced human cells.

Animals↗
PubMed1982

Greek (A gamma) variant of hereditary persistence of fetal haemoglobin: globin gene organization and studies of expression of fetal haemoglobins in clonal erythroid cultures.

Individuals heterozygous for the Greek (A gamma) variant of hereditary persistence of fetal haemoglobin (HPFH) synthesize Hb F whose gamma-globin chains are predominantly of the A gamma type. DNA obtained from Greek HPFH heterozygotes was used to test for abnormalities in the organization of non alpha-globin genes. In addition, gamma- and beta-globin expression was studied in BFUe cultures. Restriction endonuclease mapping showed that the G gamma, delta and beta genes in cis to the Greek HPFH determinant are intact. Overproduction of gamma-globin chains synthesis was observed in the BFUe cultures. A significant portion of the gamma chain synthesis was of the G gamma type, suggesting that the G gamma genes cis and trans to the HPFH chromosome are active in culture. DNA mapping data indicate that in contrast to G gamma A gamma HPFH and the G gamma (delta beta) thalassaemia, the Greek (A gamma) HPFH is not due to a large deletion in the non-alpha globin gene region. It is possible that the anomaly may result either from a small deletion or point mutation which influences non alpha-globin transcription. The in vitro synthesis data suggest that the low level of G gamma-globin chain synthesis in vivo is not the result of transcriptional inactivation of the G gamma gene, since this gene appears to be expressed in erythroid cell cultures. We speculate that the genetic lesion in Greek (A gamma) HPFH is in regulatory sequences which control the level of G gamma and A gamma expression during development.

Adult↗
PubMed1982

The organization, structure, and in vitro transcription of Alu family RNA polymerase III transcription units in the human alpha-like globin gene cluster: precipitation of in vitro transcripts by lupus anti-La antibodies.

We have studied the location, structure, and in vitro transcription of repetitive DNA sequences within the human alpha-like globin gene cluster. At least eight different Alu family repeats were identified, each of which is transcribed in vitro to produce discrete RNA transcripts. The nucleotide sequence of one Alu repeat sequence, located on the 3' side of the alpha l globin gene (3'-alpha l), was determined and compared to published Alu repeat sequences. In vitro transcription of this repeat sequence generates RNA fragments of approximately 410, 260, 160, and 86 nucleotides. To determine whether these transcripts associate with specific proteins in vitro, we carried out immunoprecipitation experiments using an antiserum from systemic lupus erythematosus (SLE) patients. We find that the antiserum anti-La, which was shown to precipitate ribonucleoproteins (RNPs) containing the adenovirus VAI RNA from virus infected cells, preferentially precipitates the smallest two in vitro transcripts of the 3'-alpha l Alu repeat. These results suggest that the RNAs interact with specific factors in the in vitro transcription reaction mix to form RNP.

Antibodies↗
PubMed1981

Identification of DNA sequences required for transcription of the human alpha 1-globin gene in a new SV40 host-vector system.

We have developed a rapid and simple method for studying the transcription of cloned eucaryotic genes, which involves transfecting SV40-transformed monkey cell lines (COS cells) with derivatives of the plasmid pBR322 that contain the SV40 viral replication origin but lack regions necessary for viral transcription (SV-ORI vectors). Because COS cells produce SV40 T antigen and are permissive for SV40 viral replication, transfected SV-ORI plasmids replicate to a high copy number. SV-ORI plasmids carrying a human alpha-globin gene are also replicated in COS cells. Moreover, the alpha-globin gene is faithfully transcribed to produce high levels of RNA, which is accurately processed to produce authentic alpha-globin mRNA. We have used this transcription system to demonstrate that a sequence located between 55 and 87 base pairs upstream from the mRNA capping site is required for efficient transcription of the alpha-globin gene in COS cells.

Animals↗
PubMed1980

Structure and in vitro transcription of human globin genes.

The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.

Base Sequence↗
PubMed1980

Molecular cloning and characterization of the human beta-like globin gene cluster.

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

Bacteriophage lambda↗
PubMed1980

The chromosomal arrangement of human alpha-like globin genes: sequence homology and alpha-globin gene deletions.

We report the isolation of a cluster of four alpha-like globin genes from a bacteriophage lambda library of human DNA (Lawn et al., 1978). Analysis of the cloned DNA confirms the linkage arrangement of the two adult alpha-globin genes (alpha 1 and alpha 2) previously derived from genomic blotting experiments (Orkin, 1978) and identifies two additional closely linked alpha-like genes. The nucleotide sequence of a portion of each of these alpha-like genes was determined. One of these sequences is tentatively identified as an embryonic zeta-globin gene (zeta 1) by comparison with structural data derived from purified zeta-globin protein (J. Clegg, personal communication), while the other sequence cannot be matched with any known alpha-like polypeptide sequence (we designate this sequence phi alpha 1). Localization of the four alpha-like sequences on a restriction map of the gene cluster indicates that the genes have the same transcriptional orientation and are arranged in the order 5'-zeta 1-phi alpha 1-alpha 2-alpha 1-3'. Genomic blotting experiments identified a second, nonallelic zeta-like globin gene (phi 2) located 10-12 kb 5' to the cloned zeta-globin gene. Comparison of the locations of restriction sites within alpha 1 and alpha 2 and heteroduplex studies reveal extensive sequence homology within and flanking the two genes. The homologous sequences, which are interrupted by two blocks of nonhomology, span a region of approximately 4 kb. This extensive sequence homology between two genes which are thought to be the products of an ancient duplication event suggests the existence of a mechanism for sequence matching during evolution. One consequence of this arrangement of homologous sequences is the occurrence of two types of deletions in recombinant phage DNA during propagation in E. coli. The locations and sizes of the two types of deletions are indistinguishable from those of the two types of deletions associated with alpha-thalassemia 2 (Embury et al., 1979; Orkin et al., 1979; S. Embury et al., manuscript submitted). This information strongly suggests that the genetic disease is a consequence of unequal crossing over between homologous sequences within and/or surrounding the two adult alpha-globin genes.

Bacteriophage lambda↗
PubMed1980

The nucleotide sequence of the human beta-globin gene.

We report the complete nucleotide sequence of the human beta-globin gene. The purpose of this study is to obtain information necessary to study the evolutionary relationships between members of the human beta-like globin gene family and to provide the basis for comparing normal beta-globin genes with those obtained from the DNA of individuals with genetic defects in hemoglobin expression.

Amino Acid Sequence↗
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↗
PubMed1980

The structure of a human alpha-globin pseudogene and its relationship to alpha-globin gene duplication.

The complete nucleotide sequence of a human alpha-globin pseudogene (psi alpha 1) is presented. Comparison of this sequence with a normal human alpha-globin gene (alpha 2) reveals that psi alpha 1 contains both an initiator codon mutation and frameshift deletions which would prevent the production of an alpha-globin polypeptide. psi alpha 1 contains two intervening sequences with sizes and locations characteristic of mammalian alpha-globin genes. However, the alteration or absence of putative splicing sequences suggests that a primary transcript of psi alpha 1 would not be processed to produce a mature mRNA. The relationship of pse alpha 1 to the adjacent duplicated alpha-globin genes alpha 1 and alpha 2 has been investigated at the nucleotide sequence level. psi alpha 1, alpha 2 and alpha 1 each possess the sequence GCCTGTGTGTGCCTG directly following their respective poly(A) addition sites. Sequence analysis of alpha 2 and alpha 1 3' flanking regions further reveals that the alpha-globin gene duplication units are exactly bordered by this homologous sequence. We discuss the possible evolutionary origin of psi alpha 1 and other globin pseudogenes in the context of globin gene duplication.

Animals↗
PubMed1980

The nucleotide sequence of a rabbit beta-globin pseudogene.

We report the nucleotide sequence of a rabbit beta-globin pseudogene, psi beta 2. A comparison the psi beta 2 sequence with that of the rabbit adult beta-globin gene, beta 1, reveals the presence of frameshift mutations and premature termination codons in the protein coding sequence which render pse beta 2 unable to encode a functional beta-globin polypeptide. psi beta 2 contains two intervening sequences at the same locations in the globin protein coding sequence as beta 1 and all other sequenced beta-globin genes. An examination of the DNA sequences at the intron/exon junctions suggests that a putative pse beta 2 precursor mRNA could not be spliced normally. We compare the flanking and noncoding sequences of pse beta 2 and beta 1 and discuss the evolutionary relationship between these two genes.

Animals↗
PubMed1980

The organization of repetitive sequences in a cluster of rabbit beta-like globin genes.

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit beta-like globin genes (beta 1-beta 4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140-400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.

Animals↗
PubMed1980

Tissue-specific DNA methylation in a cluster of rabbit beta-like globin genes.

The relationship between DNA methylation and differential expression of rabbit beta-like globin genes was studied by using restriction enzymes that cleave the sequence C-C-G-G but are differentially inhibited by the presence of 5-methylcytosine. The methylation frequency of 13 C-C-G-G sites that flank a set of four closely linked rabbit beta-like globin genes was determined. This analysis revealed that certain sites surrounding embryonic and adult globin genes are relatively undermethylated in DNA from embryonic and adult erythroid tissues, respectively. This pattern is most pronounced for three sites that are undermethylated in erythroid cells but are totally methylated in nonerythroid cells. We conclude that the degree of CpG methylation in the rabbit beta-like globin gene cluster is correlated with gene activity, but the effect is confined to relatively small regions of DNA.

Animals↗