The Putative Oncogene Pim-1 in the Mouse: Its Linkage and Variation among t Haplotypes

Pim-1, a putative oncogene involved in T-cell lymphomagenesis, was mapped between the pseudo-alpha globin gene Hba-4ps and the alpha-crystallin gene Crya-1 on mouse chromosome 17 and therefore within the t complex. Pim-1 restriction fragment variants were identified among t haplotypes. Analysis of restriction fragment sizes obtained with 12 endonucleases demonstrated that the Pim-1 genes in some t haplotypes were indistinguishable from the sizes for the Pim-1b allele in BALB/c inbred mice. There are now three genes, Pim-1, Crya-1 and H-2 I-E, that vary among independently derived t haplotypes and that have indistinguishable alleles in t haplotypes and inbred strains. These genes are closely linked within the distal inversion of the t complex. Because it is unlikely that these variants arose independently in t haplotypes and their wild-type homologues, we propose that an exchange of chromosomal segments, probably through double crossingover, was responsible for indistinguishable Pim-1 genes shared by certain t haplotypes and their wild-type homologues. There was, however, no apparent association between variant alleles of these three genes among t haplotypes as would be expected if a single exchange introduced these alleles into t haplotypes. If these variant alleles can be shown to be identical to the wild-type allele, then lack of association suggests that multiple exchanges have occurred during the evolution of the t complex.     

A major testicular cell protein specified by a mouse T/t complex gene.

The technique of two-dimensional gel electrophoresis was used to identify a major testicular cell protein, p63/6.9, which is specified by a gene (p63) within the mouse T/t complex on chromosome 17. A wild-type gene causes the expression of one form of this protein, p63/6.9b. All lethal and semilethal t haplotypes derived from wild mice cause the expression of an apparently identical alternate allelic form of the p63/6.9 protein. This protein, p63/6.9a, represents the first t haplotype-specific molecule to be biochemically identified. A dominant haplotype (T^Hp) acts as a null allele of the p63 gene; this unique behavior provides additional evidence for the interpretation of T^Hp as a deletion within the T/t region of chromosome 17. Limited proteolysis of viable testicular cells causes selective cleavage of the p63/6.9 proteins, relative to other detergent-soluble testicular cell proteins known to be internal. This result strongly suggests that p63/6.9 proteins are located on the cell surface. Qualitative and quantitative estimates indicate that p63/6.9 is one of the most prominent proteins on the testicular cell surface. p63/6.9 is expressed in all other mouse cell types analyzed but at greatly reduced levels. Partial t haplotypes obtained from infrequent recombination events were used to map the p63 gene close to the dominant mutation T and separate from the lethal factors of t haplotypes. A 100% correlation was observed between the expression of p63/6.9a and the genetic presence of the tail interaction factor of t haplotypes. The significance of this correlation in terms of the evolution of t haplotypes among wild mice is discussed.     

Genetic structure and origin of t haplotypes of mice, analyzed with H-2 cDNA probes

We investigated the genetic organization and evolutionary origin of t chromosomes of mice by examining the restriction fragment patterns of DNA from t haplotypes and normal chromosomes with cDNA probes to H-2 class I genes. On genomic DNA blots, the restriction fragments containing H-2-related sequences were highly variable among different inbred strains of mice, whereas they were very similar among different t haplotypes even when the t haplotypes carried serologically different H-2 haplotypes. These observations suggest that all t haplotypes have a common origin and are not products of independent mutational events. We also mapped the position of several restriction fragments characteristic of t DNA by using a battery of recombinant t haplotypes, defined with respect to their t-lethal factors and H-2 haplotypes. We thus show that restriction fragments containing H-2-related sequences map to the left of the H-2 class I genes in t chromosomes, a region in which the tw32 b-lethal factor also maps. The cloning of these fragments can be expected to provide an entry for the structural analysis of t DNA.     

Sixteen newH-2 haplotypes derived from wild mice

Wild mice captured in Texas, Scotland, Federal Republic of Germany, Denmark, Spain, Greece, Israel, Egypt, and Chile were mated to inbred strains and through successive backcross matings and H-2 typing lines homozygous for wild-derived H-2, haplotypes were established. The lines, which are neither congenic nor inbred, were then typed with antibodies defining known H-2 alleles at class I and class II loci. In addition, antisera were produced by the immunization of inbred strains with tissues of the new lines. Sixteen of the lines were characterized in this manner. The characterization resulted in the identification of 16 new H-2 haplotypes, 11 new K alleles, 10 new D alleles, and 21 new class I antigenic determinants, most of them of the private type. Most of the haplotypes represent natural recombinants sharing segments of the H-2 complex with previously identified haplotypes. A number of haplotypes are recombinants between the K and the A loci, which in genetic studies have proved difficult to separate. The lines, however, also provide evidence for preservation of blocks of genes in the H-2 complex, particularly in the class II region. Some of class I alleles previously found in wild mice from Michigan have now been found again in these mice. Several class II alleles of these lines appear to be the same as those found in inbred strains. Identical or nearly identical class I and class II alleles thus commonly occur in different populations. These findings strengthen the argument that in populations, H-2 alleles are relatively stable.     

A survey of susceptibility to infection with Trichinella spiralis of inbred mouse strains sharing common H-2 alleles but different genetic backgrounds

Twenty-eight different inbred strains of mice representing five different H-2 haplotypes were compared for degree of susceptibility to a primary infection with Trichinella spiralis. Marked differences in susceptibility, measured by the average number of muscle larvae per host, were seen among strains of mice sharing common H-2 alleles. The genes controlling these differences must therefore map at loci outside the major histocompatibility complex. Strains of mice sharing the H-2k haplotype were generally more susceptible than strains expressing other haplotypes and strains expressing H-2q alleles were most resistant. Strains of mice were ranked in order of decreasing susceptibility. Knowledge of these ranking may be of value to researchers wishing to select strains of mice appropriate for studies on T. spiralis.     

Genetic analysis of the proximal portion of the mouse t complex: evidence for a second inversion within t haplotypes

Genomic sequences derived from the mouse t complex by a microdissection cloning technique have been used as tools to obtain high resolution genetic maps of the wild-type and t haplotype forms of the most proximal portion of chromosome 17. Genetic mapping was performed through a recombinant inbred strain analysis and an analysis of partial t haplotypes. The accumulated data demonstrate the existence of a large inversion of genetic material, encompassing the loci of T and qk, within the proximal portion of t haplotypes. This newly described proximal inversion and the previously described distal inversion provide an explanation for the suppression of recombination observed along the length of t haplotype DNA in heterozygous mice.     

Assignment of the TCP1 locus to the long arm of human chromosome 6 by in situ hybridization

TCP1, the human homolog of the Tcp-1 locus in the mouse, which is part of the murine t complex and codes for an abundant testicular germ-cell protein, has been mapped within the human genome by in situ hybridization. Using a cDNA probe for TCP1, pB1.4 hum, we assigned TCP1 to human chromosome region 6q23----qter, with the most likely localization being 6q25----q27.     

Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time.

The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.     

Genetic Exchange across a Paracentric Inversion of the Mouse T Complex

Mouse t haplotypes are distinguished from wild-type forms of chromosome 17 by four nonoverlapping paracentric inversions which span a genetic distance of 20 cM. These inversion polymorphisms are responsible for a 100-200-fold suppression of recombination which maintains the integrity of complete t haplotypes and has led to their divergence from the wild-type chromosomes of four species of house mice within which t haplotypes reside. As evidence for the long period of recombinational isolation, alleles that distinguish all t haplotypes from all wild-type chromosomes have been established at a number of loci spread across the 20-cM variant region. However, a more complex picture emerges upon analysis of other t-associated loci. In particular, "mosaic haplotypes" have been identified that carry a mixture of wild-type and t-specific alleles. To investigate the genetic basis for mosaic chromosomes, we conducted a comprehensive analysis of eight t complex loci within 76 animals representing 10 taxa in the genus Mus, and including 23 previously characterized t haplotypes. Higher resolution restriction mapping and sequence analysis was also performed for alleles at the Hba-ps4 locus. The results indicate that a short tract of DNA was transferred relatively recently across an inversion from a t haplotype allele of Hba-ps4 to the corresponding locus on a wild-type homolog leading to the creation of a new hybrid allele. Several classes of wild-type Hba-ps4 alleles, including the most common form in inbred strains, appear to be derived from this hybrid allele. The accumulated data suggest that a common form of genetic exchange across one of the four t-associated inversions is gene conversion at isolated loci that do not play a role in the transmission ratio distortion phenotype required for t haplotype propagation. The implications of the results pose questions concerning the evolutionary stability of gene complexes within large paracentric inversions and suggest that recombinational isolation may be best established for loci residing within a short distance from inversion breakpoints.     

Molecular cloning and sequence analysis of a haploid expressed gene encoding t complex polypeptide 1

The mouse t haplotypes show defects in spermatogenesis attributed to multiple loci on chromosome 17. We have cloned the gene for an abundant testicular germ cell protein, t complex polypeptide 1, which has a variant form in t haplotypes, TCP-1A. A cDNA clone, pB1.4, which hybridizes to a 19S mRNA that is abundant in haploid cells during mouse spermatogenesis, derives from the 3' end of the mRNA encoding TCP-1B. The Tcp-1 gene appears to be a member of a novel gene family and shows multiple changes between the predicted amino acid sequences of TCP-1B and TCP-1A. An additional Taq1 site is created by a T to C transition in the predicted open reading frame of the Tcp-1a gene. The resultant RFLP has allowed typing of the Tcp-1 gene cluster in 54 complete and partial t haplotype chromosomes. DNA sequence comparison of the Tcp-1 genes suggests that the t haplotype chromosome arose within the genus Mus more than one million years ago.     

Genes in the first and fourth inversions of the mouse t complex synergistically mediate sperm capacitation and interactions with the oocyte

The t haplotypes (t) are recent evolutionary derivatives of an alternate form of the mouse t complex region located at the proximal end of chromosome 17. This variant form of approximately 1% of the mouse genome is a source of mutations altering numerous sperm functions crucial for fertilization. Males that carry two t haplotypes (t/t) are invariably sterile. t haplotypes contain four inversions relative to the wild-type t complex (+), so that in matings involving a +/t heterozygote, t is usually transmitted as a single unit. However, rare recombinants have been recovered, which carry only part of the t genotype and express only some of the t-dependent phenotypes. Use of these partial t haplotypes in genetic crosses has resulted in the general location of the two major t male sterility factors, S1 and S2, within inversions 1 and 4, respectively. Since sterility can result from a plethora of sperm defects, we have made a detailed study of various functional parameters of sperm from mice carrying S1 or S2 heterozygously or homozygously or in combination. Both S1 and S2 contain mutations altering sperm functions, including motility, capacitation, binding to the zona pellucida, binding to the oocyte membrane, and penetration of the zona pellucida-free oocyte. Therefore it seems clear that each of these factors contains multiple genes contributing to sterility. Furthermore, our results indicate that genes within S1 interact with genes in S2 for all sperm functions examined. However, S1 and S2 genes affecting motility interact in a purely additive fashion, while S1 and S2 genes affecting most other sperm characteristics interact in a synergistic manner. Additionally, the patterns of synergism between S1 and S2 for abnormalities in capacitation, sperm-oolemma binding, and zona-free oocyte penetration are nearly identical. This suggests that these three defects are caused by mutation of the same gene within each sterility factor. These findings will not only be instrumental in matching the various t haplotype sperm defects to candidate genes for S1 and S2, but will facilitate a more comprehensive understanding of the cellular and genetic mechanisms underlying t haplotype male sterility.     

Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16.

There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant.     

Multiple divergent haplotypes express completely distinct sets of class I MHC genes in zebrafish

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines; however, histocompatibility in zebrafish remains poorly understood. We examined a set of diverse zebrafish class I major histocompatibility complex (MHC) genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching has been shown to improve engraftment after hematopoietic transplantation. Using flanking gene polymorphisms, we identified six distinct chromosome 19 haplotypes. We describe several novel class I U lineage genes and characterize their sequence properties, expression, and haplotype distribution. Altogether, ten full-length zebrafish class I genes were analyzed, mhc1uba through mhc1uka. Expression data and sequence properties indicate that most are candidate classical genes. Several substitutions in putative peptide anchor residues, often shared with deduced MHC molecules from additional teleost species, suggest flexibility in antigen binding. All ten zebrafish class I genes were uniquely assigned among the six haplotypes, with dominant or codominant expression of one to three genes per haplotype. Interestingly, while the divergent MHC haplotypes display variable gene copy number and content, the different genes appear to have ancient origin, with extremely high levels of sequence diversity. Furthermore, haplotype variability extends beyond the MHC genes to include divergent forms of psmb8. The many disparate haplotypes at this locus therefore represent a remarkable form of genomic region configuration polymorphism. Defining the functional MHC genes within these divergent class I haplotypes in zebrafish will provide an important foundation for future studies in immunology and transplantation.     

Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae

Colonization of the human small intestine by Vibrio cholerae requires the type 4 toxin co-regulated pilus (TCP). Genes encoding the structure and biogenesis functions of TCP are organized within an operon located on the Vibrio Pathogenicity Island (VPI). In an effort to elucidate the functions of proteins involved in TCP biogenesis, in frame deletions of all of the genes within the tcp operon coding for putative pilus biogenesis proteins have been constructed and the resulting mutants characterized with respect to the assembly and function of TCP. As a result of this analysis, we have identified the product of one of these genes, tcpF, as a novel secreted colonization factor. Chromosomal deletion of tcpF yields a mutant that retains in vitro phenotypes associated with the assembly of functional TCP yet is severely attenuated for colonization of the infant mouse intestine. Furthermore, we have determined that the mechanism by which TcpF is translocated across the bacterial outer membrane requires the TCP biogenesis machinery and is independent of the type II extracellular protein secretion (EPS) system. These results suggest a dual role for the TCP biogenesis apparatus in V. cholerae pathogenesis and a novel mechanism of intestinal colonization mediated by a soluble factor.     

The major histocompatibility complex: the value of extended haplotypes in the analysis of associated immune diseases and disorders

Major histocompatibility complex antigens are critical to an animal's immune response. In most animals, the extreme polymorphism of MHC molecules complicates studies of the role of this complex in the immune response. In mice, however, MHC haplotype-homozygous inbred strains have been developed which are invaluable in the study of the immune system and the search for immune response genes. The human MHC bears many similarities to its murine equivalent with regard to antigen structure and polymorphism; furthermore, a number of combinations of specific MHC alleles between HLA-B and HLA-DR/DQ (extended haplotypes) are found in people more commonly than predicted by individual allele frequencies. Over 30 percent of Caucasian haplotypes are extended haplotypes, and over 55 percent of individuals have at least one extended haplotype. Examples of the same extended haplotype, even in unrelated individuals, should either all have or lack any gene within the MHC region. The value of considering extended haplotypes in searching for associations between the MHC and diseases, or immune response, is shown in three examples: congenital adrenal hyperplasia, hepatitis B immunization, and transfusion-associated graft-versus-host disease.     

Assignment of the Human Homologue of the mTRiC-P5 Gene (TRICS) to Band 1q23 by Fluorescence in Situ Hybridization

The TCP1 ring complex (TRiC) is a molecular chaperone involved in actin and tubulin folding. Little is known about the components of this complex. The first component identified was TCP1, a protein coded by a gene in the t -complex locus on mouse chromosome 17. This locus is involved in several embryonic defects, male sterility, and the transmission ratio distortion. In humans, the t-complex genes map to chromosome 6. Other components of TRiC are thought to be TCP1-related proteins. Recently, a mouse cDNA coding for one of these proteins has been cloned and named mTRiC-P5. Here we report the cloning of a partial human cDNA clone, homologous to mTRiC-P5, and its chromosome localization by fluorescence in situ hybridization. The human TRiC-P5 gene (TRIC5) maps to human chromosome 1q23, a region known to be a preferential chromosomal breakpoint involved in leukemia. Therefore, even if TCP1 and TRiC-P5 are related proteins and are found in the same protein complex, they are not coded by syntenic genes in humans.     

A mouse-based strategy for cyclophosphamide pharmacogenomic discovery.

Genome-wide mapping approaches are needed to more fully understand the genetic basis of chemotherapy response. Because of technical and ethical limitations, cancer pharmacogenomics has not yet benefited from traditional robust familial genetic strategies. We have therefore explored the use of the inbred mouse as a genetic model system in which to study response to the cytotoxic agent cyclophosphamide. Multiple phenotypes have been assessed in response to cyclophosphamide in up to 19 inbred mouse strains, including in vitro hematopoietic progenitor cell toxicity and the mobilization of hematopoietic progenitor cells into peripheral blood. Hematopoietic progenitor cell toxicity in vitro varied 2-fold among strains, whereas in vivo progenitor cell mobilization varied almost 75-fold among strains. Males mobilized more hematopoietic progenitor cells than did females, and the low-mobilization phenotype was dominant to the high-mobilization phenotype in F1 hybrid animals. In an initial attempt to analyze candidate genes, genetic variation was assessed in three cytochrome P-450 genes involved in the metabolism of cyclophosphamide. Resequencing of eight strains identified 26 polymorphisms in these genes that may influence response to cyclophosphamide. Distinct regions of high- and low-polymorphism rates were identified, and two common haplotypes were shared among the strains for each gene that exhibited variation. This phenotypic and genotypic variation among inbred strains provides a framework for cyclophosphamide pharmacogenomic discovery.     

Qualitative and quantitative variability in different classes of proteins: Comparison of mouse and rat

Summary Proteins of membranes and cytosols were extracted from the livers and brains of mice (inbred strain DBA/6J) and rats (inbred strain DA/Han) and separated by two-dimensional electrophoresis (2-DE). The 2-DE patterns were compared with regard to qualitative (spot position) and quantitative (spot intensity) characteristics of the proteins of these two species.The following results were obtained: (1) Brain had more (higher percentage) conservative proteins (proteins found in both mice and rats) than liver; (2) plasma membranes had more conservative proteins than the cytosols; (3) organ-unspecific proteins contained more conservative proteins than relatively organ-specific proteins; (4) the pattern of distribution of genetic variability among different classes of proteins represented by findings 1–3 was the same for the qualitative and quantative characteristics of the proteins; and (5) some observations indicated that quantitative variability occurred more frequently among proteins than did qualitative variability. Our conclusion is that regulatory sequences in the DNA (regulatory genes) are subjected to functional constraints that differ in strength among different classes of proteins by the same ratios as the constraints acting on the structural genes. The overall effect of the selective pressure is, however, less stringent for regulatory genes than for structural genes.The results obtained here by comparing two different species are very similar to previous results we obtained by studying different subspecies (inbred strains of the mouse). From this finding arises a new concept: the study of molecular evolution on the basis of different classes of proteins.Our results were compared with data from the literature that were obtained in part from studies on cultured cells. The comparison suggested that cultured cells have lost their tissue-specific proteins, and so generate predominantly extremely conservative proteins.