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.     

Genetic variation in mouse apolipoprotein A-IV due to insertion and deletion in a region of tandem repeats.

We have detected three unique apolipoprotein A-IV (apoA-IV) charge isoforms in strains of commensal mice. The cDNA sequences for one representative of each isoform (Mus domestesticus strains C57BL/6J and 129/J and Mus castaneus) revealed a polymorphism within a series of four imperfect repeats encoding the sequence Glu-Gln-Ala/Val-Gln. Insertions or deletions of 12 nucleotides within this repetitive region have given rise to three genotypes characterized by three (129), four (C57BL/6), or five (M. castaneus) copies of the repeat unit. To ascertain the extent of this variation among other species of the Mus genus, we sequenced this region of apoA-IV cDNAs from eight additional M. domesticus inbred strains and from five wild-derived Mus species. All eight additional M. domesticus strains examined had four repeat units, as found in C57BL/6. Among wild-derived mice, however, one species (Mus spretus) had three repeats, two species (Mus cookii and Mus cervicolor) had four repeats, and two species (Mus hortulanus and Mus minutoides) had five repeats. A lack of correlation between the number of repeat units and the phylogeny of Mus species indicates that independent mutations may have occurred throughout the evolution of specific mouse lineages. We suggest that the repetitive nature of the polymorphic sequence may predispose this region to slippage errors during DNA replication, resulting in frequent deletion/insertion mutations.     

Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.     

Variation in mouse major urinary protein (MUP) genes and the MUP gene products within and between inbred lines

The mouse major urinary proteins (MUPs) and the unprocessed in vitro translation products of MUP mRNA were each resolved by isoelectric focusing (IEF). The urinary MUPs showed about 15 distinct components, and the unprocessed MUPs about 20. In each case wide variation was observed in the relative intensities of individual bands. A comparison of three inbred lines (C57BL, BALB/c and JU) showed inter-line variation in the patterns both of the urinary MUPs and of the unprocessed MUPs. A series of experiments was carried out with a cloned MUP cDNA probe. All three inbred lines contain the same number (about 20) of MUP genes per haploid genome. In Southern blot analysis of genomic DNA the MUP genes displayed complex patterns which we interpret as showing variation on a common basic MUP gene sequence. For each combination of restriction enzymes tested, one size of fragment carried more than half of the total label, and this fragment was always the same in the three inbred lines. Inter-line differences were observed in the patterns of some of the less reactive fragments. MUP mRNA consists of at least two distinct species with sizes of 1 and 1.2 kb, which reacted with the probe in a label ratio of about 0.5 to 1. In the three inbred lines this ratio was essentially the same.     

Biosynthesis of the major urinary proteins in mouse liver: A biochemical genetic study

By labeling liver protein in vivo with [³H]leucine, the relative biosynthetic rate has been measured for the major urinary proteins (MUPs), three closely related, androgen-regulated proteins that are synthesized in mouse liver, secreted into the bloodstream, and excreted into the urine. In livers from females of strain C57BL/6J, total MUP synthesis represents about 0.6–0.9% of the total protein synthesis; in males and testosterone-treated females of the same strain, synthesis increases to about 3.5–4.0% of the total. This 4-to 6-fold induction of total MUP synthesis is similar to the androgen-mediated increase in MUP-specific messenger RNA reported by others, and indicates that the previously observed 20- to 25-fold induction of total MUP excretion into urine is generated partly at the posttranslational level. By measuring the ratio of synthesis of the individual MUPs, it was determined that the testosterone-mediated change in the relative levels of the MUPs in urine reflects a similar change in the pattern of MUP synthesis, indicating that the posttranslational processes operate on the quantity, and not the nature, of MUPs excreted. A survey of seven inbred mouse strains revealed polymorphism for the rate of total MUP synthesis in untreated females. Two classes could be distinguished on the basis of a 3- to 5-fold difference in the rate. This variation does not correlate with variation at Mup-a, a locus that controls the ratio of the three MUPs in urine from androgen-induced mice. These findings are consistent with the notion that MUP expression is controlled by a variety of independently assorting genes.     

Identification and characterization of functional genes encoding the mouse major urinary proteins.

Mouse Ltk- cells were stably transfected with cloned genes encoding the mouse major urinary proteins (MUPs). C57BL/6J MUP genomic clones encoding MUP 2 (BL6-25 and BL6-51), MUP 3 (BL6-11 and BL6-3), and MUP 4 (BL6-42) have been identified. In C57BL/6J mice, MUP 2 and MUP 4 are known to be synthesized in male, but not female, liver, and MUP 3 is known to be synthesized in both male and female liver and mammary gland. A BALB/c genomic clone (BJ-31) was shown to encode a MUP that is slightly more basic than MUP 2 and was previously shown to be synthesized in both male and female liver of BALB/c but not C57BL/6 mice. Comigration on two-dimensional polyacrylamide gels of the MUPs encoded by the transfecting gene provides a basis for tentative identification of the tissue specificity and mode of regulation of each gene. DNA sequence analysis of the 5' flanking region indicates that the different MUP genes are highly homologous (0.20 to 2.40% divergence) within the 879 base pairs analyzed. The most prominent differences in sequence occur within an A-rich region just 5' of the TATA box. This region (from -47 to -93) contains primarily A or C(A)N nucleotides and varies from 15 to 46 nucleotides in length in the different clones.     

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, L1C105, 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.     

Segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine IFN-beta gene to the murine IFN-alpha genes.

Southern blot analysis with murine (Mu) interferon (IFN)-alpha cDNA of restricted genomic DNA of three inbred strains of mice belonging to the species Mus musculus domesticus (BALB/c, C57BL/6, and DBA/2) revealed only a limited degree of polymorphism. For example, with HindIII there were only two polymorphic bands out of 14 hybridizing fragments. With Mu IFN-beta cDNA there was no polymorphism at all between BALB/c and C57BL/6 in DNA restricted with seven different enzymes. In contrast, HindIII-restricted DNA of an inbred strain of wild mice (M. spretus Lataste) hybridized with the IFN-alpha probe displayed a high degree of polymorphism compared with the three strains of laboratory mice and was also polymorphic when probed with IFN-beta cDNA. Although M. musculus domesticus and M. spretus Lataste represent different species, certain interspecies crosses are possible in the laboratory. This enabled us to follow segregation of restriction fragment length polymorphism in HindIII-restricted DNA obtained from 18 backcross progeny of a (DBA/2 X M. spretus)F1 X DBA/2 interspecies cross. There was complete coincidence between the segregation of parental (DBA/2) and (DBA/2 X M. spretus)F1-type IFN-beta and IFN-alpha restriction fragment length polymorphism, indicating tight linkage of the IFN-beta and IFN-alpha genes. In addition, in 15 of 18 progeny the segregation coincided with that of the brown locus on chromosome 4, in accord with previous results obtained with the IFN-alpha probe in strains derived from crosses between BALB/c and C57BL/6 mice. Thus, the Mu IFN-beta gene is tightly linked to the Mu IFN-alpha gene cluster on chromosome 4 near the brown locus.     

Subfamilies of the mouse major urinary protein (MUP) multi-gene family: sequence analysis of cDNA clones and differential regulation in the liver

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.     

Mapping of alpha-spectrin on distal mouse chromosome 1

DNAs from different strains of inbred mice and feral Mus spretus were found to exhibit restriction fragment length polymorphisms (RFLP) when hybridized with a probe prepared from a c-DNA clone of the mouse alpha-spectrin (Spna-1) gene. Studies of five recombinant inbred strains and (C57BL/6 X M. spretus) F1 X C57BL/6 backcross mice demonstrated that these RFLPs were allelic and that Spna-1 is closely linked to Ly-9 and Ly-17 on the distal region of chromosome 1.     

DNA Sequence Analysis of Sry Alleles (Subgenus Mus) Implicates Misregulation as the Cause of C57bl/6j-Y(pos) Sex Reversal and Defines the Sry Functional Unit

The Sry (sex determining region, Y chromosome) open reading frame from mice representing four species of the genus Mus was sequenced in an effort to understand the conditional dysfunction of some M. domesticus Sry alleles when present on the C57BL/6J inbred strain genetic background and to delimit the functionally important protein regions. Twenty-two Sry alleles were sequenced, most from wild-derived Y chromosomes, including 11 M. domesticus alleles, seven M. musculus alleles and two alleles each from the related species M. spicilegus and M. spretus. We found that the HMG domain (high mobility group DNA binding domain) and the unique regions are well conserved, while the glutamine repeat cluster (GRC) region is quite variable. No correlation was found between the predicted protein isoforms and the ability of a Sry allele to allow differentiation of ovarian tissue when on the C57BL/6J genetic background, strongly suggesting that the cause of this sex reversal is not the Sry protein itself, but rather the regulation of SRY expression. Furthermore, our interspecies sequence analysis provides compelling evidence that the M. musculus and M. domesticus SRY functional domain is contained in the first 143 amino acids, which includes the HMG domain and adjacent unique region (UR-2).     

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.     

A Single Genetic Determinant that Prevents Sex Reversal in C57BL-YPOS Congenic Mice

Sex determination in the mammalian embryo begins with the activation of a gene on the Y chromosome which triggers a cascade of events that lead to male development. The mechanism by which this gene, designated SRY in humans and Sry in mice (sex determining region of the Y chromosome), is activated remains unknown. Likewise, the downstream target genes for Sry remain unidentified at present. C57BL mice carrying a Y chromosome from Mus musculus musculus or molossinus develop normally as males. In contrast, C57BL/6 mice with the Y chromosome from M. m. domesticus often show sex reversal, i.e., develop as XY females. It has been documented that C57BL mice with the Y chromosome from Poschiavinus (Y^POS), a domesticus subtype, always develop as females or hermaphrodites. This suggests that a C57BL gene either up- or downstream of Sry is ineffective in interacting with Sry, which then compromises the processes that lead to normal male sex development. Nonetheless, by selective breeding, we have been able to generate a sex reversal-resistant C57BL/6-congenic strain of mice in which the XY^POS individuals consistently develop as normal males with bilateral testes. Because the resistance to sex reversal was transferred from strain 129S1/Sv (nonalbino) by simple selection over 13 backcross generations, it is inferred that a single autosomal gene or chromosomal region confers resistance to the sex reversal that would otherwise result. XY^POS normal males generated in these crosses were compared to XY^POS abnormal individuals and to C57BL/6 controls for sexual phenotype, gonadal weight, serum testosterone, and major urinary protein (MUP) level. A clear correlation was found among phenotypic sex, MUP level, and testis weight in the males and in the incompletely masculinized XY^POS mice. The fully masculinized males of the congenic strain resemble C57BL/6 males in the tested parameters. DNA analysis confirmed that these males, in fact, carry the Y^POS Sry gene.     

Segregation distortion of X-linked marker genes in interspecific crosses between Mus musculus and M. spretus

An interspecific cross was made between females of the C3H/HeHa.Pgk-1 a inbred laboratory strain of Mus musculus and males of the separate species Mus spretus. The F1 males are sterile but the F1 females are fertile and they were backcrossed to both C3H and spretus males. Evidence is presented from the segregation of X-linked marker genes that the interspecific F1 female has a genetically deleterious effect on the C3H X chromosome that is expressed as a male-lethal effect with the spretus Y chromosome but not with the musculus Y chromosome of C3H.     

Genes that modify expression of major urinary proteins in mice.

A survey of major urinary proteins (MUPs) from eight BALB/c mouse substrains by isoelectric focusing identified a common pattern with about 10 protein bands in males. One substrain, BALB/cJPt, differed in that it expressed two variant MUP patterns, designated 4.1lo and null. To find the chromosomal location of the gene which determines the 4.1lo phenotype, BALB/cJPt-MUP-4.1lo was crossed with a wild-derived Mus musculus domesticus inbred strain (CLA) that expresses the common BALB/c MUP pattern. The F1 phenotype revealed that the gene(s) controlling the MUP-4.1lo trait was recessive. A restriction fragment polymorphism between these strains found with a MUP cDNA probe allowed us to establish that a gene determining the MUP-4.1lo trait was not linked to the MUP structural genes on chromosome 4. Assays for other chromosomal marker loci revealed that a gene determining the MUP-4.1lo trait, designated Mupm-1, was closely linked to Myc-1 on chromosome 15. To determine the genetic basis of the null trait, BALB/cJPt-MUP-null mice were crossed with BALB/cJPt-MUP-4.1lo mice. A MUP restriction fragment polymorphism between these two lines was tightly linked to a gene or genes involved in determining the MUP-null phenotype. The two variant MUP phenotypes in BALB/cJ mice are determined by separate genes, one of which is located on chromosome 4 and the other on chromosome 15. The chromosomal location of Mupm-1 suggests that it produces a trans-acting factor which regulates MUP expression.     

Hst-3: an X-linked hybrid sterility gene

A gene, Hst-3, responsible for sterility in F1 males from crosses between Mus spretus and laboratory strains of mice such as C57BL/6, has been localized on the distal part of the X chromosome, using both DNA probes and biochemical markers on a panel of F1(C57BL/6 x SEG) x C57BL/6 backcross males. This gene may be a model for studying mammalian hybrid sterility.     

Altered Turnover of Hypoxanthine Phosphoribosyltransferase in Erythroid Cells of Mice Expressing Hprt a and Hprt b Alleles

We have previously shown that mice expressing Hprt a allele(s) have erythrocyte hypoxanthine phosphoribosyltransferase (HPRT) levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than in mice that express the Hprt b allele (Mus musculus domesticus; C57BI/6J; C3H/HeHa), and that these differences in erythrocyte HPRT levels are due to differences in the turnover rates of the HPRT A and B proteins as reticulocytes mature to erythrocytes. We show here that: the taxonomic subgroups of the genus Mus are essentially monomorphic for the occurrence of either the Hprt a or the Hprt b allele, with Hprt a being common in the aboriginal species (M. spretus, Mus hortulanus and Mus abbotti) and in several commensal species (Mus musculus musculus, M. m. castaneus, Mus musculus molossinus), while Hprt b is common in feral M. m. domesticus populations as well as in all inbred strains of mice tested; in all these diverse Mus subgroups there is a strict association of Hprt a with high and Hprt b with low levels of erythrocyte HPRT; and, the association between the occurrence of the Hprt a allele and elevated erythrocyte HPRT levels is retained following repeated backcrosses of wild-derived Hprt a allele(s) into the genetic background of inbred strains of mice with the Hprt b allele. Collectively, these observations indicate that the elevated and low levels of erythrocyte HPRT are specified by differences in the Hprt a and b structural genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evolutionary switch in tissue-specific gene expression. Abundant expression of alpha 1-antitrypsin in the kidney of a wild mouse species

alpha 1-Antitrypsin (AT), one of the major proteinase inhibitors in mammalian serum, is generally considered to be synthesized exclusively in the liver. We have found that a wild-derived Mus species, Mus caroli, expresses AT mRNA in kidney at levels approaching that in liver; no other mouse, inbred or wild-derived, exhibits this striking property. Liver and kidney mRNAs from M. caroli encode very similar AT polypeptides that are distinct from that encoded by Mus musculus liver mRNA. In vivo, liver AT is secreted into the bloodstream, while kidney AT, which is processed differently from the liver protein, is excreted into the urine. Analysis of RNA from a hybrid between M. musculus and M. caroli indicates that a cis-acting genetic element may be responsible for the difference in AT expression. Restriction enzyme digestion patterns of AT genomic sequences in M. caroli DNA are considerably different from those in M. musculus; in addition, these sequences are undermethylated in liver DNA from M. musculus and in liver and kidney DNA from M. caroli, reflecting the respective patterns of expression. Further studies of the altered tissue specificity of AT expression that is apparent in these two related species should lead to new insights into the nature and evolution of genetic determinants of tissue-specific phenotypes.