Nucleotide sequence for the htpR gene from Citrobacter freundii.
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Amino Acid Sequence↗
Biomedical subjects
S C Hardies
Publications and source records attributed to S C Hardies.
Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. beta-Cys325 is a nonessential active site residue.
Chemical modification experiments have shown that sulfhydryl groups play an important role in the mechanism of action of Escherichia coli succinyl-CoA synthetase. One of these sulfhydryl groups has been localized in the beta-subunit of the enzyme using the coenzyme A affinity analog, CoA disulfide-S,S-dioxide (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4943). Recently, it has been shown that the reactive sulfhydryl group resides in Cys325 (Nishimura, J. S., Mitchell, T., Ybarra, J., and Matula, J. M., submitted to Eur. J. Biochem. for publication). In the present study, we have changed Cys325 to a glycine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is 83% as active as wild type enzyme. In contrast to wild type succinyl-CoA synthetase, the mutant is refractory to chemical modification by CoA disulfide-S,S-dioxide and methyl methanethiolsulfonate. It is also less reactive with N-ethylmaleimide. Thus, beta-Cys325 is a nonessential active site residue.
Binding Sites↗
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↗
The L1 family in mice.
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Amino Acid Sequence↗
The potential use of repetitive sequences in phylogenetic reconstructions.
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Animals↗
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↗
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↗
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↗
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↗
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↗
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↗
Two mouse early embryonic beta-globin gene sequences. Evolution of the nonadult beta-globins.
We have determined the complete nucleotide sequence of two early embryonic beta-globin genes of the BALB/c mouse: beta h0 and beta h1 X beta h1 codes for the embryonic z protein, while the beta h0 gene may be a minor early embryonic beta-globin gene. The general sequence organization of both genes is entirely analogous to other functional globin genes. There is, however, a 220-base pair insertion of unique sequence within the first intron of beta h0 X beta h0 and beta h1 are 96% homologous for 260 base pairs 5' to the AUG initiation codon, and 93% homologous throughout their coding regions. Analysis of the 5'-flanking sequence demonstrates that these genes are more nonadult-like than adult-like. The sequences show evidence for gene conversions among the mouse nonadult beta-globin genes that were limited to individual exons, presumably by the presence of non-homologous introns. We propose that this arrangement has the beneficial evolutionary effect of allowing gene conversion to act independently on regions of the protein with different structural or functional responsibilities. beta h0 and beta h1 are evolutionary homologs to the human fetal and rabbit beta 3 genes, while their manner of expression is similar to rabbit beta 3 and dissimilar to human fetal expression. The evolutionary history of the human beta-globin genes, therefore, includes the recruitment of an embryonic gene to fetal developmental control.
Animals↗
Evolution of the mammalian beta-globin gene cluster.
We have examined relationships among the adult beta-globin genes and pseudogenes of human, rabbit, goat, and mouse. This was done by looking at homology in noncoding and flanking regions which have not been as heavily affected by gene conversion as have the coding regions of these genes. The 3' half of the beta-globin clusters of human, rabbit, and goat all originated from a two-gene ancestral cluster. We refer to the two genes as proto-delta and proto-beta after their descendants in the human genome. We propose that the 3' half of the mouse cluster originated from a four-gene ancestral cluster consisting of a proto-delta, a proto-beta, a second proto-delta, and a second proto-beta. The delta-beta intergene distance is similar among these mammals, except where altered by a 5-kilobase insertion sequence in the goat. The nature of major length differences in the large intervening sequence was also examined. In descendants of proto-beta, this region has tended to increase in length by the insertion of sequence elements about 250 base pairs long. In contrast, major length changes in the large intervening sequence of descendants of proto-delta have been mediated by expansion or contraction of a short repetitive sequence, presumably involving unequal crossovers. Possible explanations for this asymmetry in behavior are explored.
Animals↗
The diagonal-traverse homology search algorithm for locating similarities between two sequences.
We present a fast computer algorithm for finding homology between two DNA sequences. It generates a two-dimensional display in which a diagonal string of dots represents a stretch of homology between the two sequences. Our algorithm performs the search very rapidly, and has no internal data storage requirement except for the sequences themselves. These characteristics make it particularly well suited for execution on microcomputers. Without slowing execution, the matching criterion can be that a specified fraction of contiguous bases must be identical. Even with gapped sequences, we have found large search windows to be surprisingly good for detecting poor homologies with nearly complete background suppression. A diagonal search pattern is used that reports the finds in a compact and logically ordered form. A simple and rapid plotting algorithm for unsophisticated printers is also reported.
Base Sequence↗
Phenotypic variation associated with molecular alterations at a cluster of thymidine kinase genes.
Genetic variation was studied in several mouse L cell lines containing tandemly repeated herpes simplex virus thymidine kinase (TK) genes introduced by DNA-mediated gene transfer. Variants were obtained after alternate positive and negative selection for TK expression. Three classes of molecular alteration are described. One class consisted of a concerted wave of hypermethylation affecting many sites in all or nearly all of the TK genes. This resulted in genetically stable TK- variants. Of five TK+ transformants from independent transfer experiments, only one, named HM, showed this class of methylation. Hypermethylation was a reproducible phenomenon in HM, yielding TK- variants after selection with either bromodeoxyuridine or acycloguanosine [Acyclovir or 9-(2-hydroxyethy-oxymethyl)guanine]. A second class of alteration consisted of methylation affecting some, but not all, genes in the cluster. This happened in all TK+ (HAT [hypoxanthine-aminopterin-thymidine]-resistant) cell lines investigated, and this second class of methylation was incapable of generating TK- variants. Neither type of methylation was accompanied by genomic rearrangements. The third class of molecular alteration was found among TK+ (HAT-resistant) back revertants of hypermethylated HM TK- derivatives. It consisted of a 10-fold amplification of the hypermethylated TK genes. Demethylation of hypermethylated HM variants was not observed. Thus, hypermethylation in this system can be compensated for by amplification but cannot be reversed.
Animals↗
beta-globin transcript found in induced murine erythroleukemia cells is homologous to the beta h0 and beta h1 genes.
RNA transcripts complementary to at least one of the four beta-globin homologous genes (beta h) are found in high concentration in the murine erythroleukemic (MEL) cell line GM979 after butyric acid induction. Hybridization data indicate that the gene expressed is Hbb-beta h0 or Hbb-beta h1, or both. The level of beta h0/1 transcripts in the MEL cell is similar to the level of adult transcripts. The Hbb-beta h0/1 transcript is about 800 nucleotides in length. In addition, there is a larger beta h0/1 transcript of the same size and relative intensity as the adult beta-globin precursor. We also report significant levels of embryonic gene Hbb-y transcripts in induced GM979 cells. We have determined that the GM979 cell line has the [Hbb]s haplotype on the basis of an examination of its globin DNA restriction pattern. An additional MEL cell line of haplotype [Hbb]d (DBA/2 line 6A11A) was examined and found to contain no significant level of Hbb-beta h0, Hbb-beta h1, Hbb-beta h2, or Hbb-y gene transcripts.
Animals↗
Computer programs to assist in high resolution thermal denaturation and circular dichroism studies on nucleic acids.
Computer programs are described that direct the collection, processing, and graphical display of numerical data obtained from high resolution thermal denaturation (1-3) and circular dichroism (4) studies. Besides these specific applications, the programs may also be useful, either directly or as programming models, in other types of spectrophotometric studies employing computers, programming languages, or instruments similar to those described here (see Materials and Methods).
Circular Dichroism↗
RPC-5 column chromatography for the isolation of DNA fragments.
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Chromatography, Ion Exchange↗