Tom Maniatis

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.

NF-κB: A lesson in family values

NF-KB and the other members of the Rel family of transcriptional activator proteins have been a focal point for understanding how extracellular signals induce the expression of specific sets of genes in higher eukaryotes. Unlike most transcriptional activators, this family of proteins resides in the cytoplasm and must therefore translocate into the nucleus to function. The nuclear translocation of Rel proteins is induced by an extraordinarily large number of agents ranging from bacterial and viral pathogens to immune and inflammatory cytokines to a variety of agents that damage cells. Remarkably, an even larger number of genes appear to be targets for the activation by Rel proteins. As a result of these properties, we are confronted with two intriguing questions: how do multiple signal transduction pathways lead to the activation of NFKB, and how does a particular inducer lead to the activation of only one or a subset of the genes targeted by NFKB? Answers to these questions require a detailed understanding of the pathways of Rel protein activation and the mechanisms by which the genes targeted by Rel proteins are turned on. Here, we review recent progress in understanding the mechanisms involved in the activation of NF-KB, the function of individual Rel family proteins, and synergistic interactions between Rel proteins and other families of transcription factors, leading to specific gene activation. The Rel and IKB Families The Rel protein family has been divided into two groups based on differences in their structures, functions, and modes of synthesis (Baeuerle and Henkel, 1994; Siebenlist et al., 1994). The first group consists of p50 (NF-KB1) and p52 (NF-KB2), which are synthesized as precursor proteins of 105 and 100 kDa, respectively. The mature proteins, which are generated by proteolytic processing, have a so-called Rel homology domain that includes DNAbinding and dimerization domains and a nuclear localization signal. The mature proteins form functional Rel dimers with other members of the family, while dimers containing the unprocessed proteins remain sequestered in the cytoplasm. The second group of Rel proteins, which includes p65 (RelA), Rel (c-Rel), RelB, and the Drosophila Rel proteins dorsal and Dif, are not synthesized as precursors. In addition to the Rel homology domain, they possess one or more transcriptional activation domains. Members of both groups of Rel proteins can form homoor heterodimers; e.g., NF-KB is a p50-p65 heterodimer. Two types of Rel protein complexes are found in the Ub Conjugation Enzymes

Transformation of mammalian cells with genes from procaryotes and eucaryotes

Abstract We have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist. The addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT. These biochemical transformants may represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population. Co-transformation experiments were therefore performed with the viral tk gene and bacteriophage ΦX174, plasmid pBR322 or the cloned chromosomal rabbit β-globin gene sequences. Tk + transformants were cloned and analyzed for co-transfer of additional DNA sequences by blot hybridization. In this manner, we have identified mouse cell lines which contain multiple copies of 4)X, pBR322 and the rabbit β-globin gene sequences. The ΦX co-transformants were studied in greatest detail. The frequency of co-transformation is high: 15 of 16 tk + transformants contain the ΦX sequences. Selective pressure was required to identify co-transformants. From one to more than fifty ΦX sequences are integrated into high molecular weight nuclear DNA isolated from independent clones. Analysis of subclones demonstrates that the ΦX genotype is stable through many generations in culture. This co-transformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells. Ligation to either viral vectors or selectable biochemical markers is not required.

The structure and evolution of the human β-globin gene family

Argiris Efstratiadis Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115 James W. Posakony, Tom Maniatis, Richard M. Lawn* and Catherine O’Connell+ Division of Biology California Institute of Technology Pasadena, California 91125 Richard A. Spritz, Jon K. DeRiel,# Bernard G. Forget and Sherman M. Weissman Departments of Genetics and Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510 Jerry L. Slightom, Ann E. Blechl and Oliver Smithies Laboratory of Genetics University of Wisconsin Madison, Wisconsin 53706 Francisco E. Baralle, Carol C. Shoulders and Nicholas J. ProudfootQ MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England Summary We present the results of a detailed comparison of the primary structure of human p-like globin genes and their flanking sequences. Among the se- quences located 5’ to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 2 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. Calcula- tion of the divergence times of individual @like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence com- parisons. In addition, the evolutionary relationship of the embryonic e-globin gene to the other human P-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 B-like globin genes during evolution.

The isolation and characterization of linked δ- and β-globin genes from a cloned library of human DNA

A cloned library of large, random embryonic human DNA fragments was constructed and screened for β-globin sequences using the cloned human β-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 δ- and β-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′-δ-β-3′. Both the δ-and β-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 β-globin genes was detected near the 5′ end of the human β-globin gene. The two independently isolated β-globin clones differ from each other by the presence of a Pst I restriction enzyme cleavage site within the large intervening sequence of the δ-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.

The nucleotide sequence of the human β-globin gene

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

The chromosomal arrangement of human α-like globin genes: Sequence homology and α-globin gene deletions

Abstract We report the isolation of a cluster of four α-like globin genes from a bacteriophage λ library of human DNA (Lawn et al., 1978). Analysis of the cloned DNA confirms the linkage arrangement of the two adult α-globin genes (α1 and α2) previously derived from genomic blotting experiments (Orkin, 1978) and identifies two additional closely linked α-like genes. The nucleotide sequence of a portion of each of these α-like genes was determined. One of these sequences is tentatively identified as an embryonic ζ-globin gene (ζ1) by comparison with structural data derived from purified ζ-globin protein (J. Clegg, personal communication), while the other sequence cannot be matched with any known α-like polypeptide sequence (we designate this sequence ψα1). Localization of the four α-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′-ζ1-ψα1-α2-α1–3′. Genomic blotting experiments identified a second, nonallelic ζ-like globin gene (ζ2) located 10–12 kb 5′ to the cloned ζ-globin gene. Comparison of the locations of restriction sites within α1 and α2 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 α-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 α-globin genes.

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

Abstract The genes encoding human embryonic (ϵ), fetal ( G γ, A γ) and adult (δ, β) β-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage λ 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 β-like polypeptides, including the embryonic ϵ-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′-ϵ-(13.3 kb)- G γ-(3.5 kb)- A γ-(13.9 kb)-δ-(5.4 kb)-β-3′. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known β-like globin genes, we have detected two other β-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A γ-δ intergenic region while the other is located 6–9 kb 5′ to the ϵ 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′- ∗∗ ϵ- ∗G γ- A γ ∗ - ∗∗ δ-β ∗ -3′. Fine structure mapping of the region located 5′ to the δ-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.

A role for the drosophila neurogenic genes in mesoderm differentiation

The neurogenic genes of Drosophila have long been known to regulate cell fate decisions in the developing ectoderm. In this paper we show that these genes also control mesoderm development. Embryonic cells that express the muscle-specific gene nautilus are overproduced in each of seven neurogenic mutants (Notch, Delta, Enhancer of split, big brain, mastermind, neuralized, and almondex), at the apparent expense of neighboring, nonexpressing mesodermal cells. The mesodermal defect does not appear to be a simple consequence of associated neural hypertrophy, suggesting that the neurogenic genes may function similarly and independently in establishing cell fates in both ectoderm and mesoderm. Altered patterns of beta 3-tubulin and myosin heavy chain gene expression in the mutants indicate a role for the neurogenic genes in development of most visceral and somatic muscles. We propose that the signal produced by the neurogenic genes is a general one, effective in both ectoderm and mesoderm.

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.