Complete nucleotide sequence of the cosmid vector pWE15A.
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Cosmids↗
Biomedical subjects
B F Koop
Publications and source records attributed to B F Koop.
Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology.
The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.
Animals↗
Organization, structure, and function of 95 kb of DNA spanning the murine T-cell receptor C alpha/C delta region.
We have analyzed the organization, structure, and function of the murine T-cell receptor C alpha/C delta region. This region spans 94.6 kb of DNA and contains the C alpha and C delta genes, as well as the V delta 5, J delta 2, and 50 different J alpha gene segments. Within this sequence we have identified 15 new J alpha gene segments, 40 new 5' RNA splice signals, and 40 new DNA rearrangement signals for the J alpha gene segments. The murine C alpha/C delta sequence contains an exceptionally high level of coding sequence with over 5.7% of the total sequence found in the exons. This is much more than that found in the beta-globin locus and the HPRT locus. Using the sequence data obtained from the C alpha/C delta region, we have designed simple assays to test for J alpha gene segment transcription and to determine the level of polymorphism for simple repeat sequences among different inbred strains of mice using the polymerase chain reaction. Furthermore, comparisons of this 95 kb of sequence with the available sequence from homologous regions of other species have led to the identification of a highly conserved sequence that is present throughout vertebrates and in the mouse binds lymphocyte-specific nuclear proteins. Comparisons of a 10-kb region, which includes the C alpha gene in human and mouse, average 66% sequence similarity. These studies support the contention that large-scale DNA sequencing projects of homologous regions of mouse and human will provide powerful new tools for studying the biology and evolution of loci such as the T-cell receptor and for identifying and posing new questions about the functions of conserved sequences.
Amino Acid Sequence↗
Large-scale and automated DNA sequence determination.
DNA sequence analysis is a multistage process that includes the preparation of DNA, its fragmentation and base analysis, and the interpretation of the resulting sequence information. New technological advances have led to the automation of certain steps in this process and have raised the possibility of large-scale DNA sequencing efforts in the near future [for example, 1 million base pairs (Mb) per year]. New sequencing methodologies, fully automated instrumentation, and improvements in sequencing-related computational resources may render genome-size sequencing projects (100 Mb or larger) feasible during the next 5 to 10 years.
Animals↗
Primate evolution at the DNA level and a classification of hominoids.
The genetic distances among primate lineages estimated from orthologous noncoding nucleotide sequences of beta-type globin loci and their flanking and intergenic DNA agree closely with the distances (delta T50H values) estimated by cross hybridization of total genomic single-copy DNAs. These DNA distances and the maximum parsimony tree constructed for the nucleotide sequence orthologues depict a branching pattern of primate lineages that is essentially congruent with the picture from phylogenetic analyses of morphological characters. The molecular evidence, however, resolves ambiguities in the morphological picture and provides an objective view of the cladistic position of humans among the primates. The molecular data group humans with chimpanzees in subtribe Hominina, with gorillas in tribe Hominini, orangutans in subfamily Homininae, gibbons in family Hominidae, Old World monkeys in infraorder Catarrhini, New World monkeys in semisuborder Anthropoidea, tarsiers in suborder Haplorhini, and strepsirhines (lemuriforms and lorisiforms) in order Primates. A seeming incongruency between organismal and molecular levels of evolution, namely that morphological evolution appears to have speeded up in higher primates, especially in the lineage to humans, while molecular evolution has slowed down, may have the trivial explanation that relatively small genetic changes may sometimes result in marked phenotypic changes.
Animals↗
Sequencing reactions in microtiter plates.
We describe a simple and inexpensive method of performing sequencing reactions for 24 single-strand M13 DNA clones in microtiter plates. To simplify elevated temperature incubations during sequencing reactions, two heating blocks were designed to accommodate microtiter plates and fit within common laboratory heating modules. With only slight modification of standard fluorescent and radioisotopic sequencing methods, the sequencing reactions for 24 clones can be done in as little as 40 minutes.
Bacteriophages↗
Establishment and characterization of a new human Burkitt's lymphoma cell line (WSU-BL).
A new human cell line, WSU-BL, was established from a malignant ascitic fluid occurring in a patient with Burkitt's lymphoma. The established line grows in a single-cell suspension with a doubling time of 19 hours and expresses L3 morphologic features by the French-American-British classification. Immunologic study revealed that WSU-BL cells express IgM-lambda both in the cytoplasm and on the surface and react with monoclonal antibodies to B-cell antigens (B1, B4, BL3, BL4, HLA-DR, and common acute lymphoblastic leukemia antigen [CALLA]). These cells are negative for T-cell and myeloid/monocyte antigens as well as Epstein-Barr virus nuclear antigen (EBNA). These results suggest that WSU-BL corresponds to an intermediate stage of B-cell differentiation. Both fresh tumor and WSU-BL cells had a hyperdiploid karyotype carrying the 8;14 chromosome translocation. Molecular studies showed that WSU-BL has a rearrangement of c-myc proto-oncogene and expresses c-myc RNA. Phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and interferon-gamma (IFN-gamma) were able to induce several phenotypic changes on WSU-BL cells. Two-dimensional gel electrophoresis of total cellular protein showed that either TPA or IFN-gamma induced both the synthesis or loss of several proteins. Analysis of the protein patterns indicated that some proteins were uniquely responsive to either TPA or IFN-gamma and others were common to both. This cell line should be valuable for future studies of cell proliferation, differentiation, and oncogenesis concerning this neoplasm.
Adult↗
Tarsius delta- and beta-globin genes: conversions, evolution, and systematic implications.
Comparisons between duplicated genes have shown that gene conversions play an important role in the evolution of multigene families. Previous comparisons have documented in the recently duplicated gamma-fetal globin genes of catarrhine primates, over 15 separate conversions affecting extensive stretches of coding and noncoding sequences. In the present study, delta- and beta- globin genes from a lower primate Tarsius syrichta, and the delta-globin gene of the Asian great ape, Pongo pygmaeus, have been isolated and sequenced. Comparisons of these sequences with other primate delta and beta sequences confirmed a previously reported conversion in an anthropoid ancestor and revealed additional conversions in basal primate, stem haplorhine, tarsier, and early lemur lineages. Conversions found between primate delta- and beta-globin genes contrast with those found in the gamma-genes in that delta-beta conversions appear much less frequently and are more restricted to regions conserved by selection (i.e. coding and 5'-regulatory sequences). These differences indicate that soon after a duplication occurs, conversions can be quite frequent and encompass extensive portions of the duplicated region. With time, sequence differences accumulate, particularly in noncoding regions, and limit both the frequency and size of the conversions. Sequences conserved by selection accumulate differences more slowly and are therefore subject to gene conversions for a longer period of time. Both unconverted and converted sequences were consistent in supporting the placement of tarsier with anthropoids.
Animals↗
A molecular view of primate phylogeny and important systematic and evolutionary questions.
Phylogenetic analysis of extensive nucleotide sequence data from primate beta-globin gene clusters elucidates the systematics and evolution of the order Primates and reveals that rates of accumulation of mutations vary by as much as a factor of seven among different primate lineages. The picture of primate phylogeny from DNA sequences clarifies many ambiguities of the morphological picture. In the molecular picture, dwarf and brown lemurs group together into superfamily Lemuroidea, Lemuroidea and Lorisoidea into suborder Strepsirhini, and Tarsius and Anthropoidea into suborder Haplorhini. The molecular picture also provides both significant evidence for a human-chimpanzee clade that narrowly excludes gorilla and overwhelming evidence for the gorilla-chimpanzee-human clade within Hominoidea. Rates of DNA sequence evolution appear to have been fastest in the early primates ancestral to Anthropoidea and next fastest on the lorisoid branch. Rates were slowest over the past 25 Myr of hominoid descent, suggesting that mechanisms lowering the mutation rate evolved in correlation with lengthened life spans.
Animals↗
Implications of the diversity of the immunoglobulin gene superfamily.
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Animals↗
Molecular phylogeny of the family of apes and humans.
The morphological picture of primate phylogeny has not unambiguously identified the nearest outgroup of Anthropoidea and has not resolved the branching pattern within Hominoidea. The molecular picture provides more resolution and clarifies the systematics of Hominoidea. Protein and DNA evidence divides Hominoidea into Hylobatidae (gibbons) and Hominidae, family Hominidae into Ponginae (orangutan) and Homininae, and subfamily Homininae into two tribes, one for Gorilla, and the other for Pan (chimpanzee) and Homo. Parsimony and maximum likelihood analyses, carried out on orthologous noncoding nucleotide sequences from primate beta-globin gene clusters, provide significant evidence for the human-chimpanzee tribe and overwhelming evidence for the human-chimpanzee-gorilla clade. These analyses also indicate that the rate of molecular evolution became slower in hominoids than in other primates and mammals.
Animals↗
Embryonic epsilon and gamma globin genes of a prosimian primate (Galago crassicaudatus). Nucleotide and amino acid sequences, developmental regulation and phylogenetic footprints.
Sequence analysis of epsilon and gamma genes and encoded globins and high-pressure liquid chromatography analysis of globin compositions in blood hemolysates obtained from embryos, fetuses and adults show that the prosimian primate Galago crassicaudatus expresses its epsilon and gamma genes only embryonically. Since rabbit, mouse and galago all have embryonic gamma genes but simian primates have fetal gamma genes, we conclude that gamma E evolved into gamma F in stem-simians. An elevated non-synonymous substitution rate characterizes this transition. The alignment of epsilon and gamma nucleotide sequences and the parsimoniously reconstructed evolutionary history of these sequences identify several anciently conserved cis-regulatory elements (phylogenetic footprints) important for gamma expression in primates and also cis-mutations which may have been involved in the recruitment of the gamma gene to a fetal program in simian primates.
Amino Acid Sequence↗
Rhesus fetal globin genes. Concerted gene evolution in the descent of higher primates.
The comparison of the nucleotide sequences of closely linked duplicated genes of higher eukaryotes has been important in the identification of molecular events that shape the evolution of mammalian genes, most notably recombinational events such as unequal crossovers and gene conversions. Toward this goal we have been comparing the nucleotide sequences of the paired gamma 1- and gamma 2-fetal globin genes from species of catarrhine primates. Previous comparisons document that, within each great ape species as in humans, the paired gamma-genes have been involved in gene conversion events. We now extend our analysis to the catarrhine superfamily Cercopithecoidea by obtaining the nucleotide sequence of the paired gamma 1- and gamma 2-genes of rhesus monkey (Macaca mulatta). The rhesus gamma 1- and gamma 2-genes diverge less from each other than from human, chimpanzee, gorilla, and orangutan gamma 1- or gamma 2-genes. This finding indicates that a species-specific gene conversion occurred between rhesus gamma 1- and gamma 2-genes. This gamma-gene conversion (labeled C14 in our series) involved at least 1898 base pairs, extending across the complete transcriptional region of the rhesus gamma-genes. C14 could have resulted from a single large conversion or several short conversion events which may have involved the (TG)n repetitive sequence element. Parsimony analysis of the enlarged body of gamma-gene sequence data also strengthens the evidence for the 14 previously suggested gamma-gene conversion events: labeled C2, C3, and C4 in Homo; C5, C6, and C7 in Pan; C8, C9, and C10 in Gorilla; C11, C12, C13 in Pongo; C1 in the stem to Homininae (the subfamily of Homo, Pan, and Gorilla) and CO in the stem of Hominidae (the family of Pongo and Homininae).
Amino Acid Sequence↗
Evolutionary and developmental aspects of two hemoglobin beta-chain genes (epsilon M and beta M) of opossum.
A series of gene duplications that began in a stem species of Mammalia and led to five developmentally regulated hemoglobin beta-chain loci (epsilon, gamma, eta, delta, and beta) in a common ancestor of eutherian orders Artiodactyla, Rodentia, Lagomorpha, and Primates had important consequences in mammalian evolution. Findings reported here indicate that two progenitors of the five linked genes existed by the time of the eutherian (placental mammal)-metatherian (marsupial mammal) split and that these two genes were already differentiated with respect to their promoter regions and developmental expression. Southern blot and sequence analyses of the hemoglobin beta-chain genes of the opossum (Didelphis virginiana) revealed only two genes, one with coding and promoter sequences similar to eutherian prenatally expressed epsilon, gamma, and eta genes and the other coding for adult opossum hemoglobin beta-chains and having eutherian adult beta-type promoters. The most parsimonious arrangement of greater than 80 beta-globin exon sequences depicts the opossum embryonic-type gene as orthologously related to eutherian epsilon, gamma, and eta genes and the opossum adult-type gene as orthologously related to delta and beta genes. These data further indicate that after the initial beta duplication in the stem of Mammalia, the locus that became developmentally delayed in its expression evolved at a faster rate than the locus that became embryonically expressed.
Amino Acid Sequence↗
Molecular systematics of higher primates: genealogical relations and classification.
We obtained 5' and 3' flanking sequences (5.4 kilobase pairs) from the psi eta-globin gene region of the rhesus macaque (Macaca mulatta) and combined them with available nucleotide data. The completed sequence, representing 10.8 kilobase pairs of contiguous noncoding DNA, was compared to the same orthologous regions available for human (Homo sapiens, as represented by five different alleles), common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). The nucleotide sequence for Macaca mulatta provided the outgroup perspective needed to evaluate better the relationships of humans and great apes. Pairwise comparisons and parsimony analysis of these orthologues clearly demonstrated (i) that humans and great apes share a high degree of genetic similarity and (ii) that humans, chimpanzees, and gorillas form a natural monophyletic group. These conclusions strongly favor a genealogical classification for higher primates consisting of a single family (Hominidae) with two subfamilies (Homininae for Homo, Pan, and Gorilla and Ponginae for Pongo).
Animals↗
Orangutan fetal globin genes. Nucleotide sequence reveal multiple gene conversions during hominid phylogeny.
We have determined the nucleotide sequences of the linked gamma 1- and gamma 2- fetal globin genes from a single orangutan (Pongo pygmaeus) chromosome and compared them with the corresponding genes of other simian primates (gamma 1- and gamma 2-genes of human, chimpanzee, gorilla, and the single gamma-gene of the spider monkey). Previous studies have indicated that the two gamma-gene loci in catarrhine primates resulted from a duplication about 25-35 million years ago. However, comparisons of aligned gamma-gene sequences show that these genes contain three regions with distinct histories of which only the 3' third clearly reflects the ancestral nature expected of the gamma-gene duplication. To explain these different evolutionary histories and also hominid relationships we provide evidence for the occurrence of sequence conversions which affect region 1 (120 base pairs 5'-flanking through exon 2) in all hominid species and extend to varying degrees into region 2 (intron 2 through exon 3). Close examinations of the proposed conversions further suggest that 12 of the 13 conversions identified involved gamma 1 converting gamma 2. Polarity of these conversions may be a result of differential survival between these genes because during human fetal development the gamma 1-gene is preferentially expressed over the gamma 2-gene and it may be subjected to greater selection pressure to remain unaltered.
Animals↗
Globins: a case study in molecular phylogeny.
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Animals↗