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M H Edgell

Publications and source records attributed to M H Edgell.

At least 37 records · Page 2Linked to original sources
PubMed1988

The F-type 5' motif of mouse L1 elements: a major class of L1 termini similar to the A-type in organization but unrelated in sequence.

It has previously been shown that the L1 family in the mouse (L1Md) contains two alternative 5' ends called the A- and F-type sequences (1,2). We show here that the F-type element is a major class of murine L1 elements and report on the details of organization of the 5' motif of these F-type elements. Although the A- and F-type 5' sequences share no detectable sequence homology the organization of an F-type 5' end is strikingly similar to that of an A-type. That is, the F-type 5' sequences consist of a tandem array of a small number of 206 bp monomers while the A-type 5' motif consists of a tandem array of 208 bp monomers. All of the A-type elements characterized to date have a truncated monomer at the 5' end of the array. Many of the F-type elements are also terminated at the 5' end by a truncated copy but unlike the A-type elements some F-type elements terminate with a monomer which is within a few nucleotides of being complete. In addition the F-type consensus sequence, in contrast to the A-type sequence, shows homology (70%) to the body of the L1Md starting at the position where the monomer joins the rest of the L1 element.

Animals↗
PubMed1988

Extensive movement of LINES ONE sequences in beta-globin loci of Mus caroli and Mus domesticus.

LINES ONE (L1) is a family of movable DNA sequences found in mammals. To measure the rate of their movement, we have compared the positions of L1 elements within homologous genetic loci that are separated by known divergence times. Two models that predict different outcomes of this analysis have been proposed for the behavior of L1 sequences. (i) Previous theoretical studies of concerted evolution in L1 have indicated that the majority of the 100,000 extant L1 elements may have inserted as recently as within the last 3 million years. (ii) Gene conversion has been proposed as an alternative to a history of prolific recent insertions. To distinguish between these two models, we cloned and characterized two embryonic beta-globin haplotypes from Mus caroli and compared them with those of M. domesticus. In 9 of 10 instances, we observed an L1 element to be present in one chromosome and absent at the same site in a homologous chromosome. This frequency is quantitatively consistent with the known rate of concerted evolution. Therefore, we conclude that gene conversion is not required for concerted evolution of the L1 family in the mouse. Furthermore, we show that the extensive movement of L1 sequences contributes to restriction fragment length polymorphism. L1 insertions may be the predominant cause of restriction fragment length polymorphisms in closely related haplotypes.

Animals↗
PubMed1987

Determination of a functional ancestral sequence and definition of the 5' end of A-type mouse L1 elements.

The complete nucleotide sequence of L1Md-A13, a 6372 base-pair (bp) member of the L1Md repetitive family isolated from a BALB/c mouse genomic DNA library, is reported. The nucleotide sequence of 4331 bp from the 5' end of L1Md-9, which is located in the beta-globin complex of the C57BL/10 mouse, is also reported. Parsimony analysis of these sequences plus two previously reported L1Md sequences allows the determination of an ancestral L1Md sequence. Analysis of the L1Md population indicates that this ancestral sequence is likely to represent a functional L1 sequence. This ancestral sequence confirms that the length (1137 bp and 3900 bp) and relationship (14 bp overlap) of the two large open reading frames previously reported are conserved features of the L1Md family. It also allows the determination of an ancestral amino acid sequence for these two open reading frames. Full-length L1Md elements have one of two sequences tandemly repeated at the 5' end. These two monomers are called A-type and F-type. Our data define the 5' end of A-type full-length L1Md elements. L1Md elements of the A-type have varying numbers of tandemly repeated 208 bp monomers, but each element ends about 78 bp from the 5' end of the terminal 208 bp monomer.

Animals↗
PubMed1987

Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli.

The ability to express the genes of pathogenic human viruses, such as the acquired immune deficiency syndrome (AIDS) virus (also called human immunodeficiency virus) in bacterial cells affords the opportunity to study proteins that are ordinarily difficult or inconvenient to obtain in amounts sufficient for detailed analysis. A segment of the AIDS virus pol gene was expressed in Escherichia coli. Expression resulted in the appearance of reverse transcriptase activity in the bacterial cell extracts. The extracts contained two virus-related polypeptides that have the same apparent molecular weights as the two processed forms of virion-derived reverse transcriptase (p66 and p51). The formation of these two polypeptides depended on the coexpression of sequences located near the 5' end of the pol gene, a region that is thought to encode a viral protease. This bacterial system appears to generate mature forms of the AIDS virus reverse transcriptase by a proteolytic pathway equivalent to that which occurs during virus infection of human cells.

DNA Restriction Enzymes↗
PubMed1987

The molecular organization of the beta-globin complex of the deer mouse, Peromyscus maniculatus.

Recombinant DNA clones have been isolated that contain 80 kb of the beta-globin complex from the deer mouse, Peromyscus maniculatus. Comparisons of this complex with that from the laboratory mouse, Mus domesticus (with an order 5'-Hbby, Hbb-bhO, Hbb-bhl, Hbb-bh2, Hbb-bh3, Hbb-bl, Hbb-b2 3') highlight organizational trends in the beta-globin complex since the two species diverged. Unlike other mammals studied thus far, the deer mouse possesses three adult genes. Partial sequence analysis indicates that each of the three adult genes is intact and hence may be functional. Hybridization of one of the two Mus pseudogenes, Hbb-bh3, to genomic blots from Peromyscus reveals that it has a homologous counterpart in Peromyscus. Homologous genes to the two gamma-like Mus genes, Hbb-bhO and Hbb-bhl, are also found in Peromyscus. The strong hybridization between the Hbb-bhl genes and significant nucleotide similarity between the Hbb-bhO genes suggest that both pairs are important for the ontogeny of these mice although no known product has been identified for the Hbb-bhO genes. The presence of Hbb-bhO and Hbb-bhl in Peromyscus suggests that the duplication that created this related gene set occurred before the two lineages diverged. A single gene for Hbb-y has been isolated from Peromyscus. The adult region in Peromyscus has undergone significant divergence from the same region in Mus, having three rather than two adult genes, the acquisition of at least 15 kb of extra DNA relative to Mus, and possibly the loss of the Hbb-bh2 pseudogene. The nonadult region of the complex, in contrast, contains the same set of genes apparently distributed over the same amount of DNA as in the Mus beta-globin complex. This observation suggests that the embryonic region of the complex is more evolutionarily stable than the adult region.

Animals↗
PubMed1986

Conservation throughout mammalia and extensive protein-encoding capacity of the highly repeated DNA long interspersed sequence one.

We report an investigation of the structure, evolutionary history, and function of the highly repeated DNA family named Long Interspersed Sequence One (L1). Hybridization studies show, first, that L1 is present throughout marsupial and placental mammalian orders. Second, L1 is more homologous within these species than between them, which suggests that it has undergone concerted evolution within each mammalian lineage. Third, on the whole L1 diverges in accordance with the fossil record. This suggests that it arose in each lineage rather by inheritance from a common ancestral family, which was present in the progenitor to mammals, than by cross-species transmission. Alignment of 1.6 X 10(3) bases of primate and mouse L1 DNA sequences shows a predominance of silent mutations within aligned long open reading frames, indicating that at least this part of L1 has produced functional protein. The observation of additional long open reading frames in further unaligned DNA sequences suggests that a minimum of 3.2 X 10(3) bases or at least half of the L1 structure is a protein-coding sequence. Thus L1, which contains about 100,000 members in mouse, is by far the most repetitive family of which a subset comprises functional protein-encoding genes. The ability of the putative protein-encoding regions of mouse L1 to hybridize to L1 homologs throughout the Mammalia implies that these sequences have been subject to conservative selection upon protein function in all mammalian lineages, rather than in a few. L1 is therefore a highly repeated family of genes with both a widespread and an ancient history of function in mammals.

Amino Acid Sequence↗
PubMed1986

A complete library of point substitution mutations in the glucocorticoid response element of mouse mammary tumor virus.

The glucocorticoid response element (GRE) of mouse mammary tumor virus (MMTV) was chemically synthesized as two complementary DNA strands bearing cohesive termini. During automated synthesis, random mutations were introduced into the DNA by "doping" each of the four nucleoside phosphoramidites (A, G, C, and T) with a low level of the other three. These preparations were annealed and cloned into an M13 phage vector to produce a library of GRE mutants. Mutations within the synthesized region were identified by sequencing phage isolates at random. All of the chemically distinct classes of transition and transversion mutations have been observed. Statistical considerations indicate that the library contains all of the possible 90 point substitution mutations within a 30-nucleotide mutagenic target. So far 88 of these substitutions have been isolated, 74 as single mutants. At least two of the three possible single mutants at each of the 30 positions have been identified.

Base Sequence↗
PubMed1986

A simple nonisotopic method for restriction mapping in single-stranded DNA cloning vectors based on taking timepoints during primed Klenow synthesis.

A fast, simple, and nonisotopic method for restriction mapping inserts in single-stranded cloning vectors (such as M13 or single-stranded plasmids) is presented. The procedure uses a commercially available oligonucleotide sequencing primer to initiate Klenow-mediated, unidirectional DNA synthesis along the single-stranded insert DNA. Aliquots taken at very short timepoints from this reaction are quick-frozen, heat-inactivated, and restriction-digested with the restriction enzyme or enzymes of interest. When the samples are run on an agarose gel and stained with ethidium bromide, the restriction bands appear in the order of their proximity to the priming site. The method's advantages are that it is fast, unidirectional and thus relatively unambiguous, requires neither isotope nor elaborate DNA handling or extraction procedures, and resolves the ambiguities due to "near doublets" that often plaque double-digest mapping and partial-digest mapping. Tetranucleotide restriction maps extending up to 5 kb can be determined from a single priming experiment; more infrequent hexanucleotide restriction sites can be mapped over longer distances. Also, a single aliquot taken at an early timepoint can be restriction-digested to establish the orientation of cloned inserts.

Cloning, Molecular↗
PubMed1986

An analysis of replacement and synonymous changes in the rodent L1 repeat family.

L1 is a family of long interspersed repetitive sequences in mammals that includes the BamHI family in rodents and the KpnI family in primates. Previous studies have shown that L1 repeats contain a long open reading frame and that the family evolves in concert. Working with 32 rodent elements for which DNA sequence is available, we used the distribution of replacement and synonymous changes to determine which L1 lineages had been expressing their reading frame. The evidence obtained is consistent with there having been a small number of L1 genes that have been expressing a functional protein. Much of the concerted evolution in L1 is accounted for by the tendency of these functioning L1 genes to continually create nonfunctional pseudogenes by reinsertion into the genome of sequences derived from their transcripts. The gain of new pseudogenes is balanced by the loss of old pseudogenes with a half-life of 2 Myr. Therefore, most of the observed L1 repeats are at a dead end with respect to either the expression of the L1 protein or the potential to elaborate further copies of themselves. However, the turnover of L1 pseudogenes is sufficient to constitute a vast flux of sequences into and then out of the flanking regions of all cellular genes. If the presence of flanking L1 pseudogenes affects the expression of other genes in even a subtle fashion, this process should represent a major source of genetic variation. A second level of concerted evolution occurs within the functional L1 sequences in a pattern that did not meet our expectations for selfish DNA. Also, in spite of the marked suppression of replacement relative to synonymous changes in functioning L1 genes, they evolve at an overall rate accelerated to the level of their own pseudogenes.

Animals↗
PubMed1986

The sequence of a large L1Md element reveals a tandemly repeated 5' end and several features found in retrotransposons.

The complete nucleotide sequence of a 6,851-base pair (bp) member of the L1Md repetitive family from a selected random isolate of the BALB/c mouse genome is reported here. Five kilobases of the element contains two overlapping reading frames of 1,137 and 3,900 bp. The entire 3,900-bp frame and the 3' 600 bp of the 1,137-bp frame, when compared with a composite consensus primate L1 sequence, show a ratio of replacement to silent site differences characteristic of protein coding sequences. This more closely defines the protein coding capacity of this repetitive family, which was previously shown to possess a large open reading frame of undetermined extent. The relative organization of the 1,137- and 3,900-bp reading frames, which overlap by 14 bp, bears resemblance to protein-coding, mobile genetic elements. Homology can be found between the amino acid sequence of the 3,900-bp frame and selected domains of several reverse transcriptases. The 5' ends of the two L1Md elements described in this report have multiple copies, 4 2/3 copies and 1 2/3 copy, of a 208-bp direct tandem repeat. The sequence of this 208-bp element differs from the sequence of a previously defined 5' end for an L1Md element, indicating that there are at least two different 5' end motifs for L1Md.

Amino Acid Sequence↗
PubMed1985

Transposition of a long member of the L1 major interspersed DNA family into the mouse beta globin gene locus.

A long member of the highly repeated long interspersed DNA family L1Md (for L1 in Mus domesticus) has integrated by transposition into a target site which lies between the two adult beta globin genes of mouse. DNA hybridization and nucleotide sequence analysis show that this target site, which is part of the single copy DNA flanking the globin genes, is interrupted by the L1 element in one chromosome but is uninterrupted in both allelic and ancestral chromosomes. Other large DNA rearrangements of the region between the two adult beta globin genes are also associated with these allelic chromosomes, and include insertions or deletions of both single copy DNA and simple and complex repetitive DNA. This has caused extensive reorganization of this intergenic region. However, the distance between the two genes flanking this region remains conserved, suggesting that the spacing of the globin genes may be subject to conservative selection.

Alleles↗
PubMed1985

An empirical method for the evaluation of the quality of genomic DNA libraries.

An empirical method is presented that enables one to determine, with only a relatively small investment of time and materials, whether a lambda genomic DNA library will be a productive source of clones carrying specific sequences. The method provides an indication of the abundance of the sequences in library DNA and indicates whether major DNA rearrangements have occurred during library construction and amplification.

Animals↗
PubMed1985

Tempo and mode of concerted evolution in the L1 repeat family of mice.

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.

Animals↗
PubMed1984

The mouse globin pseudogene beta h3 is descended from a premammalian delta-globin gene.

The beta h3 pseudogene of the BALB/c mouse contains sequence defects which prevent transcription and translation to produce a beta-globin. Comparison with other globin gene sequences indicates that beta h3 arose by recombination between an adult beta-globin gene and some significantly diverged globin sequence. Analysis of noncoding sequences shows that the 3' end of mouse beta h3 and the human delta-globin gene are both descended from an ancestral gene, which we call proto-delta. The origin of proto-delta must predate the mammalian radiation. A member of the L1 family of interspersed repetitive elements is inserted into the 3' untranslated delta-homologous sequence in beta h3 from BALB/c. beta h3 is a widespread feature of the rodent beta-globin complex, which has been fixed in the genome for 35 million years. Independent inactivation events produced pseudogenes located between the adult and nonadult beta-globin genes in the rodent, primate, rabbit, and goat lineages. One model to explain the abundance and evolutionary persistence of pseudogenes postulates that the mammalian genome simply has no efficient mechanism for deleting nonessential sequences. Consequently, the genomes of higher eukaryotes have been growing, by the accumulation of duplications, with doubling times of 200 +/- 100 million years.

Animals↗
PubMed1984

Dispersal process associated with the L1 family of interspersed repetitive DNA sequences.

We have determined the complete nucleotide sequence for five members of the L1Md repetitive family from the beta-globin gene region of the BALB/c mouse. The five repeats are different lengths, each terminating at the 5' end at different points with respect to one another. We have analyzed the nucleotides around the endpoints of the five repeats for clues as to the mechanisms involved with the dispersal and 5' truncation of this repeat family. Each L1 member is flanked by a pair of short direct repeats. Since these direct repeats differ in length and sequence in each of the five cases, the dispersal mechanism does not involve a sequence targeted process. The sequence at the 3' end is conserved and its organization resembles the 3' end of a polyadenylated RNA, suggesting that transcripts of the repeat are involved in the dispersal process either directly or as intermediates in the generation of complementary DNA copies of the sequence. One of the L1 repeats is a recent insertion, since it is found in the Hbbd chromosome, but not in the Hbbs chromosome. This suggests a dispersal process that has been active as recently as 4 million years ago.

Animals↗
PubMed1984

The complete nucleotide sequence of a beta-globin-like structure, beta h2, from the [Hbb]d mouse BALB/c.

We have determined the complete nucleotide sequence of beta h2, a pseudogene in the mouse beta-globin gene complex. The structure of beta h2 is analogous to that of a normal beta-globin gene, and its nucleotide sequence shares 72% homology with the coding regions of a reference mouse adult beta-globin gene. A frame shift occurs in the first coding region for which a compensatory splicing scheme can be devised. The reading frame is not otherwise disrupted. All of the recognized transcription, translation, and splicing signals in beta h2 are intact, with the exception of the " CCAAT box," which has been altered to GTAAC . We compared the predicted amino acid sequence of beta h2 with other beta-globin sequences. Evidence for a period of divergence without selection in the history of beta h2 was found in a set of codons that are usually highly conserved in productive beta-globin genes. An evolutionary tree constructed from nucleotide sequence suggests that beta h2 originated from the adult genes at least 60 million years ago. After some period as a productive gene, beta h2 was inactivated and has subsequently diverged without selection. Hybridization experiments demonstrated that beta h2 and the surrounding region occur without major alteration in other rodent species. The sequence ( AGCCA - 4n - GTGT ) occurs 5' of the CCAAT box in beta h2 and in many productive globin genes.

Animals↗
PubMed1984

The mouse beta h1 gene codes for the z chain of embryonic hemoglobin.

Beta h0 and beta h1 are two beta-like globin genes in the mouse beta-globin gene cluster whose functions have not previously been established. Transcripts of both beta h0 and beta h1 are found in yolk sac-derived erythroid cells from mouse embryos and in the murine erythroleukemic cell line GM979. S1 nuclease analysis shows that beta h1 is the more abundant of the two transcripts in both embryonic erythroid cells and the cell line GM979. Hybridization of a beta h1-specific probe to RNA from erythroid cells of 10-, 11-, 12-, 13-, and 14-day-old mouse embryos indicates that levels of beta h1 mRNA are high at 10 and 11 days and then decline during further fetal development. The in vitro translation product of hybrid-selected beta h1 mRNA was analyzed by acid/urea polyacrylamide gel electrophoresis. The beta h1 translation product has the electrophoretic mobility expected for the z chain. We conclude that beta h1 is a fully functional gene which codes for the embryonic z chain.

Animals↗