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.

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.

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.

Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese.

Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33,000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.

The dynamics of murine LINE-1 subfamily amplification.

We have examined the dynamics of replication of the mouse LINE-1 retrotransposon within a single large expanding LINE-1 family found in a particular population of Mus spretus. This family has reached thousands of copies per haploid genome within 0.1 to 0.2 Myr and accounts for most, if not all, LINE-1 replication since 0.4 Myr ago. The family shows only one split into two clades during this time. From these data we propose a model that links the evolution of LINE-1 to the dynamics of its migration among mouse populations. We hypothesize that selected LINE-1 elements, referred to as master sequences, each amplify a subfamily within a distinct mouse population before migrating into the global mouse population. When these master sequences come in contact with each other by migration, generally one continues to expand at the expense of the other. We further discuss potential tests of this model.

Systematic identification of LINE-1 repetitive DNA sequence differences having species specificity between Mus spretus and Mus domesticus.

LINE-1 is a family of repetitive DNA sequences interspersed among mammalian genes. In the mouse haploid genome there are about 100,000 LINE-1 copies. We asked if the subspecies Mus spretus and Mus domesticus have developed species-specific LINE-1 subfamilies. Sequences from 14 M. spretus LINE-1 elements were obtained and compared to M. domesticus LINE-1 sequences. Using a molecular phylogenetic tree we identified several differences shared among a subset of young repeats in one or the other species as candidates for species-specific LINE-1 variants. Species specificity was tested using oligonucleotide probes complementary to each putative species-specific variant. When hybridized to genomic DNAs, single-variant probes detected an expanded number of elements in the expected mouse. In the other species these probes detected a smaller number of matches consistent with the average rate of random divergence among LINE-1 elements. It was further found that the combination of two species-specific sequence differences in the same probe reduced the detection background in the wrong species below our detection limit.

Shared sequence variants of Mus spretus LINE-1 elements tracing dispersal to within the last 1 million years.

LINE-1 repetitive sequences contain a record of an evolving population of transposons within the mammalian genome. Of the 100,000 copies of LINE-1 sequences per genome there are many shared sequence variants representing changes occurring within the propagating LINE-1 elements themselves, rather than changes that occur during retrotransposition or after an element inserts in the genome. These shared sequence variants define families of LINE-1 elements which have spread within specific periods of time. We have been interested in studying events in LINE-1 evolution since the speciation of Mus spretus and Mus domesticus approximately 3 million years (Myr) ago. To do this, we have collected LINE-1 sequences that have shared sequence variants specific to M. spretus. The sampled LINE-1 elements were sequenced at their extreme 3' ends, where the density of sequence variants is highest. The new sequences define six new M. spretus-specific sequence variants. Of these, we have found one that could be used to screen for LINE-1 elements arising in the last 1 Myr, which we argue is a critical sample for understanding the dynamics of LINE-1 propagation.

Mus spretus LINE-1s in the Mus musculus domesticus inbred strain C57BL/6J are from two different Mus spretus LINE-1 subfamilies.

A LINE-1 element, LIC105, was found in the Mus musculus domesticus inbred strain, C57BL/6J. Upon sequencing, this element was found to belong to a M. spretus LINE-1 subfamily originating within the last 0.2 million years. This is the second spretus-specific LINE-1 subfamily found to be represented in C57BL/6J. Although it is unclear how these M. spretus LINE-1s transferred from M. spretus to M. m. domesticus, it is now clear that at least two different spretus LINE-1 sequences have recently transferred. The limited divergence between the C57BL/6J spretus-like LINE-1s and their closest spretus ancestors suggests that the transfer did not involve an exceptionally long lineage of sequential transpositions.

Mus spretus LINE-1 sequences detected in the Mus musculus inbred strain C57BL/6J using LINE-1 DNA probes.

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.

Preparation and characterization of large amounts of restriction fragments containing the E. coli lac control elements.

Large quantities of pure DNA fragments (789, 203 and 95 bp in length) containing the Escherichia coli lac controlling elements (operator, promoter, CRP binding site) were prepared from appropriate recombinant plasmids. High pressure liquid chromatography on RPC-5 or preparative sucrose gradient centrifugation was used to fractionate the pVH51 vector from the inserts. The fragments had few, if any, nicks or depurinated sites, and the majority of the fragment ends were intact. Absorbance-temperature profiles on the fragments showed multiphasic transitions.

Sequencing and enhanced expression of the gene encoding diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A) phosphorylase in Saccharomyces cerevisiae.

The gene, DTP, coding for diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A) phosphorylase was isolated from a Saccharomyces cerevisiae genomic DNA library in lambda gt11. In yeast and Escherichia coli transformed with the multicopy vector, YEp352, containing the cloned DTP gene, the Ap4A phosphorylase was produced at levels nine- to 17-fold higher than in untransformed hosts. The nucleotide (nt) sequence was determined. The gene codes for a polypeptide chain of 321 amino acids (aa). Two-aa sequence motifs of possible significance were identified: a potential adenine nt binding site and a potential phosphorylation site. The DTP gene is located on yeast chromosome III and is present as a single copy. Although multicopy vector expression increased the Ap4A phosphorylase activity ninefold above the endogenous activity in transformed yeast, the intracellular concentration of Ap4A did not decrease and the growth rate of the yeast was unchanged.

Temporal and topological clustering of diverged residues among enterobacterial dihydrofolate reductases.

The complete nucleotide and encoded amino acid sequences were determined for the dihydrofolate reductase (DHFR) from the bacteria Enterobacter aerogenes and Citrobacter freundii. These were compared with the closely related Escherichia coli DHFR sequence. The ancestral DHFR sequence common to these three species was reconstructed. Since that ancestor there have been seven, nine, and one amino acid replacements in E. coli, E. aerogenes, and C. freundii, respectively. In E. coli, five of its seven replacements were located in the beta-sheet portion of the protein, and all seven were located in a single restricted region of the protein. In E. aerogenes, all nine of its replacements were located within surface residues, with five clustered in a region topologically distinct from the E. coli cluster. The replaced side chains are sometimes in direct contact but more often are separated by an intervening side chain. It is argued that the temporal clustering of replacements is typical for the evolution of most proteins and that the associated topological clustering gives a picture of how evolutionary change is accommodated by protein structure.

LINE-1 repetitive DNA probes for species-specific cloning from Mus spretus and Mus domesticus genomes.

Mus domesticus and Mus spretus mice are closely related subspecies. For genetic investigations involving hybrid mice, we have developed a set of species-specific oligonucleotide probes based on the detection of LINE-1 sequence differences. LINE-1 is a repetitive DNA family whose many members are interspersed among the genes. In this study, library screening experiments were used to fully characterize the species specificity of four M. domesticus LINE-1 probes and three M. spretus LINE-1 probes. It was found that the nucleotide differences detected by the probes define large, species-specific subfamilies. We show that collaborative use of such probes can be employed to selectively detect thousands of species-specific library clones. Consequently, these probes could be exploited to monitor and access almost any given species-specific region of interest within hybrid genomes.

Preparative fractionation of DNA restriction fragments by reversed phase column chromatography.

Reversed phase column chromatography on RPC-5 resin was used to fractionate milligram quantities of DNA fragments generated by restriction endonucleases. Fractionation was on the basis of size, the presence or absence of sticky ends, and at least one as yet undertermined property.

Targeted cloning of a subfamily of LINE-1 elements by subfamily-specific LINE-1-PCR.

Subfamily-specific LINE-1 PCR (SSL1-PCR) is the targeted amplification and cloning of defined subfamilies of LINE-1 elements and their flanking sequences. The targeting is accomplished by incorporating a subfamily-specific sequence difference at the 3' end of a LINE-1 PCR primer and pairing it with a primer to an anchor ligated within the flanking region. SSL1-PCR was demonstrated by targeting amplification of a Mus spretus-specific LINE-1 subfamily. The amplified fragments were cloned to make an SSL1-PCR library, which was found to be 100-fold enriched for the targeted elements. PCR primers were synthesized based on the sequence flanking the LINE-1 element of four different clones. Three of the clones were recovered from Mus spretus DNA. A fourth clone was recovered from a congenic mouse containing both Mus spretus and Mus domesticus DNA. Amplification between these flanking primers and LINE-1 PCR primers produced a product in Mus spretus and not in Mus domesticus. These dimorphisms were further verified to be due to insertion of Mus spretus-specific LINE-1 elements into Mus spretus DNA and not into Mus domesticus DNA.

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.

Identification of regions of pRZ2 which have delayed elution behavior on RPC-5 column chromatography.

DNA restriction fragments generally elute from RPC-5 in order of their size. However, some fragments elute later than predicted. Chromatographic studies were performed on five different restriction digests (Hae III, Hha I, Alu I, Taq I, and Hae III + HindII) of pRZ2 DNA in an effort to localize the regions which have the delayed properties. Also, the magnitude of the delay was quantitated in each case. Most of the delayed fragments were localized in one major (931 bp) and one minor (approximately 210 bp) region of the genome. The fragments exhibiting a greater extent of delay were in the major region. The results described herein and in the following paper show that, in most cases, this effect can be explained by the base composition, or sequence of the fragments, or both.

Gene flow of unique sequences between Mus musculus domesticus and Mus spretus.

Allelic diversity has been examined from a variety of Mus musculus subspecies and Mus spretus strains by sequencing at a 453-bp unique sequence locus. One M. m. domesticus classic inbred strain, C57BL/KsJ, contained a sequence identical to that in the M. spretus wild-derived inbred strain SEG, and other wild M. spretus isolates. Such a result should have been precluded by the expected divergence between the species unless there has been interspecies gene flow. Examination of C57BL/KsJ for M. spretus-specific repetitive sequences shows that it is neither a mis-identified spretus strain nor a domesticus/spretus hybrid. Thus, in addition to the previously reported presence of small amounts of Mus spretus-specific repetitive DNA in M. m. domesticus, there is a detectable flow of unique sequence between the two species. There was also ancestral polymorphism observed among the spretus alleles. The difficulty of distinguishing ancestral polymorphism from horizontal transfer is discussed.