A Repeated Segment on the Mouse Y Chromosome Is Composed of Retroviral-Related, Y-Enriched and Y-Specific Sequences

We report the isolation and characterization of two recombinant clones containing DNA derived from the Y chromosome of the C57BL/10 inbred mouse strain. Both clones were isolated from a lambda phage library derived from a partial EcoRI digest of C57BL/10 male DNA using the murine retrovirus M720. Characterization of these clones showed they were derived from a repeated segment present on the C57BL/10J Y chromosome that contains sequences found elsewhere in the genome. In addition, one clone contained a sequence, designated YB10, that is unique to the Y chromosome and present in approximately 500 copies on the C57BL/10J Y chromosome. Analysis of Southern blots containing DNAs prepared from females and males of representative species from four subgenera of Mus probed with pYB10 and the 3'LTR from one of the Y-associated retroviruses (MuRVY) revealed that, with the exception of a single fragment observed in both female and male DNA of Mus saxicola, hybridization to pYB10 was observed only to male DNA of the species Mus spretus, Mus hortulanus, Mus musculus, Mus domesticus and Mus abbotti. In addition, the pattern and intensity of hybridization to YB10 and the MuRVY-LTR indicated that sequence of divergence was followed by amplification of Y chromosome sequences containing YB10 and MuRVY. The divergence and amplification occurred separately in each of the ancestral lineages leading to M. spretus, M. hortulanus, M. abbotti, M. musculus and M. domesticus. We suggest that acquisition and amplification of DNA sequences by the mammalian Y chromosome has contributed to its evolution and may imply that the mammalian Y chromosome is evolving at a faster rate than the rest of the genome.     

Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus

A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046- related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another.      

The amplified long genomic sequence (ALGS) located in the centromeric regions of mouse chromosomes.

We reported previously that the haploid genome of standard strains of laboratory mice contains approximately 70 copies of an amplified long genomic sequence, designated ALGS, that includes a retroposon of the gene for elongation factor 2 (MER). The length of each repeating unit is more than 60 kb, and the sequence of the unit is highly conserved among the repeats. In the present study, Southern blot analysis of the genomes of wild rodents demonstrated that the ALGS is present in all subspecies of Mus musculus and is abundant in M. spicilegus, whereas it is absent in M. spretus as well as in Rattus and other closely related genera. This result indicates that the amplification occurred after the species differentiation with the genus Mus and at least prior to the differentiation of subspecies of M. musculus. To locate chromosomal positions of the ALGS, in situ hybridization was carried out with laboratory strains and wild mice. It appears that the ALGS is located in the centromeric regions of most chromosomes in laboratory mice, M. musculus and M. spicilegus, whereas no positive signals were observed with M. spretus, in accordance with the results from the Southern blotting analysis.     

Y Chromosome Evolution in the Subgenus Mus (Genus Mus)

A 305 base pair DNA sequence isolated from the Y chromosome of the inbred mouse strain C57BL/10 was used to investigate the pattern and tempo of evolution of Y chromosome DNA sequences for five species in the subgenus Mus, including Mus spretus, Mus hortulanus, Mus abbotti, Mus musculus and Mus domesticus. Variation in hybridization patterns between species was characterized by differences in fragment lengths of both intensely and faintly hybridizing fragments, whereas variation in hybridization patterns within species was characterized primarily by differences in fragment lengths of faintly hybridizing fragments. Phylogenetic analyses were conducted based on fragment size variation within and among species. Phylogenetic relationships inferred from these analyses partly agree with the phylogenetic relationships obtained from biochemical and mitochondrial DNA data. We conclude that a set of DNA sequences common to the Y chromosomes of a closely related group of species in the subgenus Mus has evolved rapidly as reflected by sequence divergence and sequence amplification.     

Isolation and Characterization of a Mouse Y Chromosomal Repetitive Sequence

The Y chromosome plays a dominant role in mammalian sex determination, and characterization of this chromosome is essential to understand the mechanism responsible for testicular differentiation. Male mouse genomic DNA fragments, cloned into pBR322, were screened for the presence of Bkm (a female snake satellite DNA)-related sequences, and we obtained a clone (AC11) having a DNA fragment from the mouse Y chromosome. In addition to a Bkm-related sequence, this fragment contained a Y chromosomal repetitive sequence. DNA isolated from the XX sex-reversed male genome produced a hybridization pattern indistinguishable to that obtained with normal female DNA, suggesting that the AC11 sequence is not contained within the Y chromosomal DNA present in the sex-reversed male genome. Based on the hybridization patterns against mouse Y chromosomal DNA, AC11 classified 16 inbred laboratory strains into two categories; those with the Mus musculus musculus type Y chromosome and those with the M.m. domesticus type Y chromosome. Three European subspecies of Mus musculus (M.m. brevirostris, M.m. poschiavinus and M.m. praetextus) possessed the M.m. domesticus type Y chromosome, whereas the Japanese mouse, M.m. molossinus, had the M.m. musculus type Y chromosome. The survey was also extended to six other species that belong to the genus Mus, of which M. spretus and M. hortulamus showed significant amounts of AC11-related sequences in their Y chromosomes. The male-specific accumulation of AC11-related sequences was not found in M. caroli, M. cookii, M. pahari or M. platythrix. This marked difference among Mus species indicates that the amplification of AC11-related sequences in the mouse Y chromosome was a recent evolutionary event.     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.     

Genetics of intracisternal-A-particle-related envelope-encoding proviral elements in mice.

Intracisternal-A-particle-related envelope-encoding (IAPE) proviral elements in the mouse genome encode and express an envelope-like protein that may allow transmission of IAPEs as infectious agents. To test IAPE mobility and potential transmission in mice, we have analyzed the distribution of IAPE elements in the genomes of Mus spretus and Mus musculus inbred strains and wild-caught animals. Potential full-length (IAPE-A) proviral elements are present as repetitive copies in DNA from male but not female animals of M. musculus inbred strains and Mus musculus castaneus. Analysis of IAPE-cellular junction fragments indicates that fixation of most IAPEs in the germ line occurred in M. musculus and M. spretus after speciation but before M. musculus inbred strains were derived.     

Geographic origin of the Y Chromosomes in “old” inbred strains of mice

Six distinct Y Chromosomes (Chr) were identified among 39 standard inbred strains of mice with five probes that identified Y Chr-specific restriction fragments on Southern blots. Three Y Chr types, distributed among 31 strains, were of Asian Mus musculus origin. The remaining three Y Chr types, distributed among eight strains, were of M. domesticus origin. The Asian source of the M. musculus Y Chr was confirmed by determining the DNA sequence of 221 bp from an open reading frame within the Sry (sex determining region Y) gene (Gubbay et al., Nature 346 245–250, 1990) in three inbred strains (C57BL/6J, AKR/J, and SWR/J) and comparing the sequence to the homologous sequences derived from wild caught European and Asian M. musculus males. These data indicate that a minimum of six male mice contributed to the formation of the old inbred strains.     

Detection of Nitrosomonas spp. by polymerase chain reaction

Abstract A unique genomic DNA fragment was isolated from Nitrosomonas europaea ATCC 19718. Based on the sequence of this fragment, oligonucleotide primers for polymerase chain reaction amplification were prepared which amplify sequences of 775 and 658 bp. The predicted DNA fragments were both amplified from the genome of N. europaea and a Nitrosomonas spp. isolated from a local oxidation pond. The primers failed to amplify DNA from the genomes of the ammonia oxidiser Nitrosolobous multiformis , the nitrite oxidiser Nitrococcus mobilis as well as from the genomes of other unrelated heterotrophic bacteria. These DNA sequences could be amplified from 0.01 ng of N. europaea genomic DNA or from 100 intact cells, and it was possible to detect Nitrosomonas DNA in a DNA mixture extracted from water samples drawn from a local oxidation pond.     

Identification and characterization of a novel conserved DNA repeat

Homozygous In(10)17Rk mice contain a paracentric inversion within Chromosome (Chr) 10 and exhibit a pygmy phenotype, suggesting that the distal inversion breakpoint is within the pygmy (pg) locus. In order to obtain the pygmy gene by positional cloning procedures, In(10)17Rk DNA was subjected to RFLP analysis with single-copy probes derived from the wild-type pygmy locus. This analysis identified a DNA polymorphism in the DBA/2J mouse strain on which the In(10)17Rk mutation was originally induced. A detailed characterization of this polymorphism revealed the presence of a novel, tandemly repeated DNA element. Copy number estimation experiments indicate that there are approximately 100,000 copies of this element in the haploid DBA/2J genome. PCR typing studies revealed the presence of the repeat at the pygmy locus of 6 of the 18 Mus domesticus strains analyzed. The absence of the repeat from the pygmy locus of 12 strains of the M. domesticus species and from the M. caroli, M. spretus, M. castaneus, and M. molossinus species suggests that the repeat could serve as a strain-specific hybridization probe in genetic mapping studies. Finally, the novel tandem DNA repeat is conserved in both rat and human genomes as indicated by Southern hybridization experiments.     

Evolution of a mouse Y chromosomal sequence flanked by highly repetitive elements

Mammalian primary sex is determined by the presence or absence of the Y chromosome. However, little is known about the molecular processes through which the Y chromosome exerts its action. We applied recombinant DNA techniques to isolate mouse Y chromosomal fragments and described previously a clone designated as AC11 (Y. Nishioka and E. Lamothe. 1986. Genetics, 113:417-432). To obtain information on DNA sequences that flank AC11, we screened a mouse genomic library for the presence of AC11-related sequences and isolated over 50 positive clones. In this report we describe clones ACC2 and ACC3, both of which contain highly repetitive elements. Using a male-specific portion of these clones, we compared DNA's isolated from mice (Mus musculus, M. hortulanus, M. spretus, M. cookii, M. pahari, and M. platythrix), rat, hamster, and guinea pig and obtained results that agree with the phylogenetic relationships deduced from morphological and biochemical studies. The male-specific accumulation of the related sequences was found only in M. musculus, M. hortulanus, and M. spretus.     

Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.     

Rolling circle amplification of genomic templates for inverse PCR (RCA–GIP): a method for 5′- and 3′-genome walking without anchoring

We have devised an improved method of genome walking, named rolling circle amplification of genomic templates for Inverse PCR (RCA–GIP). The method is based on the generation of circular genomic DNA fragments, followed by rolling circle amplification of the circular genomic DNA using ϕ29 DNA polymerase without need for attachment of anchor sequences. In this way from the circular genomic DNA fragments, after RCA amplification, a large amount of linear concatemers is generated suitable for Inverse PCR template that can be amplified, sequenced or cloned allowing the isolation of the 3′- and 5′- of unknown ends of genomic sequences. To prove the concept of the proposed methodology, we used this procedure to isolate the promoter regions from different species. Herein as an example we present the isolation of four promoter regions from Crocus sativus, a crop cultivated for saffron production.     

Whole genome amplification and sequencing of a Daphnia resting egg

Resting eggs banks are unique windows that allow us to directly observe shifts in population genetics, and phenotypes over time as natural populations evolve. Though a variety of planktonic organisms also produce resting stages, the keystone freshwater consumer, Daphnia, is a well‐known model for paleogenetics and resurrection ecology. Nevertheless, paleogenomic investigations are limited largely because resting eggs do not contain enough DNA for genomic sequencing. In fact, genomic studies even on extant populations include a laborious preparatory phase of batch culturing dozens of individuals to generate sufficient genomic DNA. Here, we furnish a protocol to generate whole genomes of single ephippial (resting) eggs and single daphniids. Whole genomes of single ephippial eggs and single adults were amplified using Qiagen REPLI‐g Single Cell kit reaction, followed by NEBNext Ultra DNA Library Prep Kit for library construction and Illumina sequencing. We compared the quality of the single‐egg and single‐individual amplified genomes to the standard batch genomic DNA extraction in the absence of genome amplification. At mean 20× depth, coverage was essentially identical for the amplified single individual relative to the unamplified batch extracted genome (>90% of the genome was covered and callable). Finally, while amplification resulted in the slight loss of heterozygosity for the amplified genomes, estimates were largely comparable and illustrate the utility and limitations of this approach in estimating population genetic parameters over long periods of time in natural populations of Daphnia and also other small species known to produce resting stages.     

Patterns of transitional mutation biases within and among mammalian genomes

Significant transition/transversion mutation bias is a well-appreciated aspect of mammalian nuclear genomes; however, patterns of bias among genes within a genome and among species remain largely uncharacterized. Understanding these patterns is important for understanding similarities and differences in mutational patterns among genomes and genomic regions. Therefore, we have conducted an analysis of 7,587 pairs of sequences of 4,347 mammalian protein-coding genes from seven species (human, mouse, rat, cow, sheep, pig, and macaque) and from the introns of 51 gene pairs and multiple intergenic regions (37 kbp, 52 kbp and 65 kbp) from the human, chimpanzee, and baboon genomes. Our analyses show that genes and regions with widely varying base composition exhibit uniformity of transition mutation rate both within and among mammalian lineages, as long as the transitional mutations caused by CpG hypermutability are excluded. The estimates show no relationship to potential intrachromosomal or interchromosomal effects. This uniformity points to similarity in point mutation processes in genomic regions with substantially different GC-content biases.     

Chloroplast genome evolution in the genus Avena

The genus Avena contains five different chloroplast genomes, I-V. A physical map of chloroplast (ct) DNA of Avena sativa (type I chloroplast genome) was constructed using three restriction endonucleases, PstI, SalI and SmaI. This genome is ca. 135.5 kbp in size, and contains two inverted repeats of ca. 22.5 kbp each, separated by a large (ca. 79.0 kbp) and small (ca. 12.5 kbp) single copy region. The rbcL gene which codes for the large subunit of ribulose 1,5-bisphosphate carboxylase, was located in the map. Restriction fragment patterns of all five chloroplast genomes were compared, and among them five fragment size and five restriction site mutations were disclosed. Four site mutations were found in two or more chloroplast genomes, the other site and five fragment size mutations were specific to one or another of the chloroplast genomes. A dendrogram showing phylogenetic relationships among the five chloroplast genomes, based on the distribution of the common and specific mutations among them, indicates that chloroplast genome divergence characterized by three restriction site mutations occurred first between two diploid groups, each carrying A and C genome (nuclear), respectively, followed by further speciation in each group.     

Evolution of a long-range repeat family in Chromosome 1 of the genus Mus

Copy numbers and variation of a clustered long-range repeat family on Chromosome (Chr) 1 have been studied in different species of the genus Mus. The repeat sequence was present in all, as inferred from cross-hybridization with probes derived from the Mus musculus repeat family. Copy numbers determined by dot blot hybridization were very low, from three to six per haploid genome in M. caroli, M. cervicolor, and M. cookii. These species form one branch of the phylogenetic tree in the genus Mus. In the other group of phylogenetically related species—M. spicilegus, M. spretus, M. musculus and M. macedonicus—copy numbers ranged from 6 to 1810 per haploid genome. The repeat cluster is cytogenetically visible as a fine C-band in M. macedonicus and as a C-band positive homogeneously staining region (HSR) in several populations of M. m. domesticus and M. m. musculus. When cytogenetically visible, the clusters contained from 179 to 1810 repeats. Intragenomic restriction fragment length polymorphisms (RFLPs), which reflect sequence variation among different copies of the long-range repeat family, increased with higher copy numbers. The high similarity of the RFLP pattern among genomes with C-band positive regions in Chr 1 of M. m. musculus, M. m. domesticus, and M. macedonicus points to a close evolutionary relationship of their Chr 1 repeat families.     

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.