The molecular genetics of human hemoglobins.
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Animals↗
Biomedical subjects
T Maniatis
Publications and source records attributed to T Maniatis.
Characterisation of deletions which affect the expression of fetal globin genes in man.
Deletions in the DNA of individuals with hereditary persistence of fetal haemoglobin (HPFH) and 8 beta-thalassaemia have been mapped as a means of identifying regulatory sequences involved in the switch from fetal to adult globin gene expression. The end points of these deletions have been precisely located with respect to restriction endonuclease cleavage sites within and surrounding the gamma-, delta- and beta-globin genes in normal human DNA and the deletion maps were used to obtain definitive evidence for the physical linkage of the fetal and adult beta-like globin genes in the order 5'Ggamma-Agamma-delta-beta 3'. Correlation of haematological data and the location of deletions in two cases of HPFH and one case of deltabeta-thalassaemia suggest that a region of DNA located near the 5'-end of the delta-globin gene may be involved in the suppression in cis of gamma-globin gene expression in adults. The interpretation of a second case of deltabeta-thalassaemia is complicated by the fact that the deletion removes the Agamma-gene in addition to the region near the 5'-end of the delta-globin gene.
Chromosome Deletion↗
Transformation of mammalian cells with genes from procaryotes and eucaryotes.
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Animals↗
The linkage arrangement of four rabbit beta-like globin genes.
Four different regions of rabbit beta-like globin gene sequences designated beta 1, beta 2, beta 3 and beta 4 were identified in a set of clones isolated from a bacteriophage lambda library of chromosomal DNA fragments (Maniatis et al., 1978). Restriction mapping and blot hybridization (Southern, 1975) studies indicate that a subset of these clones containing beta 1 and beta 2 hybridizes to an adult beta-globin cDNA clone (Maniatis et al., 1976) more efficiently than to a human gamma-globin cDNA clone (Wilson et al., 1978), while another subset containing beta 3 and beta 4 displays the converse hybridization specificity. beta 1 was identified as the adult beta-globin gene, while beta 2, beta 3 and beta 4 have not been identified with any known rabbit globin polypeptides. Cross-hybridization and transcriptional orientation experiments indicate that the set of beta-like gene clones contains overlapping restriction fragments encompassing 44 kb of rabbit chromosomal DNA. In addition, all four genes have the same transcriptional orientation and are arranged in the order 5'-beta 4-beta 3-beta 2-beta 1-3'.
Animals↗
The structure and transcription of four linked rabbit beta-like globin genes.
Rabbit chromosomal DNA contains a cluster of four linked beta-like globin genes arranged in the orientation 5'-beta 4-(8kb)-beta 3-(5 kb)-beta 2-(7-kb)-beta 1-3'. Determination of the nucleotide sequence of gene beta 1 confirms that this gene corresponds to the second type of two common co-dominant alleles encoding the adult beta-globin chain. With the exception of two nucleotide substitutions in the large intervening sequence (intron), the intron and flanking sequences are identical with the nucleotide sequence of the first type determined by Weissmann et al. (1979). A 14S polyadenylated transcript containing large intron sequences (possibly a mRNA precursor) is detected in the bone marrow cells of anemic rabbits. Gene beta 2 has limited sequence homology to adult and embryonic beta-globin probes and lacks a detectable mRNA transcript in the erythropoietic tissues examined. It contains at least one intervening sequence analogous to the large intron in gene beta 1. Genes beta 3 and beta 4 both contain an intron of 0.8 kb. Partial DNA sequence analysis indicates that the large intron in beta 4 is located between codons for amino acids lysine and leucine in an analogous position to that of the large intron in beta 1. In addition, a second smaller intron interrupts the 5' coding sequences of gene beta 4. Both genes beta 3 and beta 4 are transcribed in embryonic globin-producing cells. Their DNA sequence homology is limited, however, to a segment of approximately 0.2 kb located on the 5' side of the large intron.
Animals↗
Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families.
A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.
Animals↗
Introduction and expression of a rabbit beta-globin gene in mouse fibroblasts.
The cloned chromosomal rabbit beta-globin gene has been introduced into mouse fibroblasts by DNA-mediated gene transfer (transformation). In this report, we examine the expression of the rabbit gene in six independent transformants that contain from 1 to 20 copies of the cloned globin gene. Rabbit globin transcripts were detected in two of these transformants at steady-state concentrations of 5 and 2 copies per cell. The globin transcripts from one cell line are polyadenylylated and migrate as 9S RNA on methylmercury gels. These transcripts reflect correct processing of the two intervening sequences but lack 48 +/- 5 nucleotides present at the 5' terminus of rabbit erythrocyte globin mRNA.
Animals↗
The isolation of structural genes from libraries of eucaryotic DNA.
We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of beta-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.
Base Sequence↗
The isolation and characterization of linked delta- and beta-globin genes from a cloned library of human DNA.
A cloned library of large, random embryonic human DNA fragments was constructed and screened for beta-globin sequences using the cloned human beta-globin cDNA plasmid pJW102 (Wilson et al., 1978) as a hybridization probe. Two independent clones were obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing. Each of the clones carries both the adult delta- and beta-globin genes. The two genes are separated by approximately 5.4 kilobases (kb) of DNA and their orientation with respect to the direction of transcription is 5'-delta--beta-3'. Both the delta- and beta-globin genes contain a large noncoding intervening sequence (950 and 900 bp, respectively) located between the codons for amino acids 104 (arginine) and 105 (leucine). Although the location of the large intervening sequence within the coding regions of the two genes is identical, the two noncoding sequences bear little sequence homology. A second, smaller intervening sequence similar to that found in other mammalian beta-globin genes was detected near the 5' end of the human beta-globin gene. The two independently isolated beta-globin clones differ from each other by the presence of a Pst I restriction enzyme cleavage site within the large intervening sequence of the delta-globin gene of one of the clones. This suggests that the human DNA carried in the two clones was derived from two homologous chromosomes which were heterozygous for the Pst I restriction enzyme recognition sequence.
DNA Restriction Enzymes↗
Studies on the structure of genes expressed during development.
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Age Factors↗
Amplification and characterization of eukaryotic structural genes.
An approach to the study of eukaryotic structural genes which are differentially expressed during development is described. This approach involves the isolation and amplification of mRNA sequences by in vitro conversion of mRNA to double-stranded cDNA followed by molecular cloning in bacterial plasmids. This procedure provides highly specific hybridization probes that can be used to identify genes and their contiguous DNA sequences in genomic DNA, and to detect specific RNA transcripts during development. The nature of the method allows the isolation of individual mRNA sequences from a complex population of molecules at different stages of development.
Animals↗
The primary structure of rabbit beta-globin mRNA as determined from cloned DNA.
The rabbit beta-globin DNA insertion of the hybrid plasmid PbetaG1 (Maniatis et al., 1976) was sequenced by the method of Maxam and Gilbert (1977). A sequence of 576 nucleotides was determined and verified by pyrimidine tract analysis of double-stranded DNA, synthesized in vitro starting from beta-globin mRNA. The derived sequence is in complete agreement with previously reported partial mRNA sequencing data and with the predictions from the primary structure of the protein. Moreover, the globin DNA insertion is missing only 13 nucleotides corresponding to the 5' terminal sequence of the mRNA. The rabbit beta-globin mRNA consists of a coding region of 438 nucleotides, flanked by a 5' noncoding region of 56 nucleotides (including the initiation codon AUG but not the 7-methyl-guanine of the "cap structure") and by a 3' noncoding region of 95 nucleotides (including a UGA termination codon). The features of the mRNA sequence are discussed with specific attention to the selective use of particular codons, the probable existence extensively base-paired segments at the 5' terminal region and the ribosome binding site. The faithful representation of beta-globin mRNA in the PbetaG1 DNA insertion establishes the validity of used cloned DNA, initially derived from double-stranded DNA transcripts of mRNA, for studying the structure of eucaryotic genes.
Animals↗
Amplification and characterization of a beta-globin gene synthesized in vitro.
Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.
Base Sequence↗
Enzymatic in vitro synthesis of globin genes.
Full-length, single-stranded rabbit globin cDNA, synthesized by AMV reverse transcriptase, apparently contains a small double-stranded sequence (hairpin) at the 3' terminus. This cDNA can serve as template-primer for E. coli DNA polymerase I, which synthesizes a strand complementary to the cDNA and covalently bound to it. The loop connecting the two strands can be cut by S1 nuclease. Reassociation, hybridization, and restriction endonuclease studies, as well as electrophoretic analyses, indicate that the sequential actions of reverse transcriptase, DNA polymerase 1, and S1 nuclease generate full-length, double-stranded synthetic globin genes.
Animals↗
A DNA operator-repressor system.
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Coliphages↗
Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.
We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.
Base Sequence↗
Recognition sequences of repressor and polymerase in the operators of bacteriophage lambda.
Nucleotide sequences in two wild-type and six mutant operators in the DNA of phage lambda are compared. Strikingly similar 17 base pair units are found which we identify as the repressor binding sites. Each operator contains multiple repressor binding sites separated by A-T rich spacers. Elements of 2 fold rotational symmetry are present in each of the sites. Superimposed on each operator is an E. coli RNA polymerase recognition site (promoter). Similarities in the sequences of the two lambda promoters, a lac promoter, and an E. coli RNA polymerase recognition site in SV40 DNA are noted.
Base Sequence↗
Full length and discrete partial reverse transcripts of globin and chorion mRNAs.
Rabbit globin mRNA was copied by AMV reverse transcriptase in the presence of various concentrations of deoxyribonucleotides (dNTPs). The cDNAs were analyzed by electrophoresis under denaturing conditions in formamide-polyacrylamide gels. Discrete size products were detected, ranging from 65 to 650 nucleotides-that is, up to the full length of the mRNA template. Increasing the concentrations of all four dNTPs stimulated formation of full-length transcripts and made the incomplete copies less abundant. Hybridization and nuclease digestion experiments indicated that the full-size product is indeed a complete transcript of globin mRNA. Similar results were obtained with chorion mRNAs. The possible usefulness of the discrete partial transcripts is discussed.
Animals↗