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In order to obtain a genomic clone of Zfy-1 from a Y chromosome of (YDOM) origin, we cloned size-fractionated SJL/J DNA in EMBL-4 and selected colonies which hybridized to pDP1007, a human zinc finger Y clone. The specificity of the clone in hybridizations to mouse and human DNA and partial sequencing confirmed that the clone (subcloned as pGZfy1D) was of Zfy-1 origin. Studies on the expression during testicular development of mRNAs hybridizing to the clone suggested that the gene is expressed post-meiotically.

Several recent studies indicate that the von Recklinghausen neurofibromatosis (NF1) gene is located near the centromere of chromosome 17 in some families. However, variable expressivity and a very high mutation rate suggest that defects at several different loci could result in phenotypes categorized as NF1. In order to assess this possibility and to map the NF1 gene more precisely, we have used two polymorphic DNA markers from chromosome 17 to screen several pedigrees for linkage to NF1. We ascertained a large Caucasian pedigree (33 individuals sampled, 17 NF1 affected) as well as eight smaller pedigrees and nuclear families (50 individuals sampled, 30 NF1 affected). Here, we report strong evidence of linkage of NF1 to the centromeric marker D17Z1 (maximum LOD = 4.42) and a weaker suggestion of linkage to the ERBA1 oncogene (maximum LOD = 0.57), both at a recombination fraction of zero. Since obligate crossovers with NF1 were not observed for either marker in any of the informative families tested, the possibility of NF1 locus heterogeneity is not supported.

The effects of the mouse major histocompatibility complex, H-2, on phospholipid methyltransferase I and II activities were investigated on hepatocyte membranes from inbred, congenic and recombinant strains. Each methyltransferase was assayed individually by measuring the incorporation of radiolabel from into endogenous phospholipids. Our results indicate that H-2 exerted a significant effect on methyltransferase I but not on methyltransferase II activity. Thus, as in lower eukaryotes, two distinct enzymes were involved in methylation of phosphotidylethanolamine (PE) to phosphatidylcholine (PC). In addition, this effect was localized to the K end of the major histocompatibility complex by the use of recombinant hepalotypes.

The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.

We have examined effects of an autosomal gene on the expression of serologically detectable H-Y, a male-specific minor histocompatibility antigen for which a role in primary sex determination has been postulated. Quantitative immunoabsorption studies using three different male specific antisera revealed variation in the level of H-Y among inbred mouse strains and mice bearing variants of the T/t complex. Two patterns of expression in inbred strains were observed; high, C57BL/6J (B6) and low, which included A/J (A), C3H/DiSn and DBA/2J. In F1 hybrids between A (female parent) and B6 (male parent), the F1 expressed H-Y levels indistinguishable from A, the low parent. Among T/t complex variants, mice bearing T or t0 showed high expression whereas those of the t12 haplotype showed low expression. The co-segregation of H-Y expression types with these T/t complex variants provided further evidence for the location of a gene determining the level of H-Y antigen on the proximal portion of chromosome 17.

It has been demonstrated that susceptibility to glucocorticoid-induced formation of cleft palate is regulated by the mouse histocompatibility complex (H-2). This has encouraged us to examine H-2 effects on glucocorticoid binding in tissues of adult animals which would provide sufficient material with which to study the biochemical mechanism of the H-2 effect. Although it has been reported that cytosol prepared from lungs of adult mice with a high susceptibility to steroid-induced cleft palate formation have a higher level of glucocorticoid binding than lung cytosol prepared from a low-susceptibility strain, we are unable to demonstrate any influence of H-2 on binding capacity in this tissue from adult animals when glucocorticoid receptors are assayed in the presence of receptor reducing and stabilizing agents that maximize binding capacity. Cytosol prepared from rat liver contains an endogenous receptor-reducing system composed of NADPH and thioredoxin. It has also been reported that the murine H-2 complex contains a gene(s) that regulates the level of a modifier(s) in fetal hepatic cytosol that affects the binding of glucocorticoids to the receptor. Of two known low molecular weight modifiers that could account for this effect, we have previously established that the heat-stable, steroid receptor "modulator" is not regulated by the H-2 complex. In the present work we have assayed thioredoxin, a second potential modifier, in liver cytosols prepared from adults of two pairs of two H-2 congenic mouse strains. Our results show that the amount of thioredoxin is the same in all four mouse strains and that it is not regulated by the H-2 locus. At this time, we are unable to identify a system in adult mice in which the widely reported regulation of glucocorticoid binding by the mouse histocompatibility locus can be submitted to definitive biochemical study.

The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [3H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.

The development of a spermatozoon (sperm) from a spermatid involves a complex process of differentiation during which a variety of new gene products appear. It has been generally assumed that no genetic transcription occurs after meiosis and, if this were so, that all the new sperm proteins would have to be transcribed from stored messenger RNA. However, the biochemical evidence suggests that there is no abrupt change in the rate of RNA synthesis during meiosis and that qualitative changes in RNA synthesis, to the extent that they are known, favor the likelihood of continuing messenger RNA synthesis. Experimental analyses of distorted transmission ratios of t-alleles and unbalanced chromosomal states in males also suggest that genes are expressed in haploid nuclei after meiosis. It is probable that spermatozoa are functionally equivalent in most respects because of intercellular bridges that create a continuous cytoplasm between developing spermatozoa, facilitating an exchange of most post-meiotic gene products. Plasma membrane proteins which are potential antigens might not be shared across the intercellular bridges but the evidence to date for haploid expression of sperm antigens is poor.

It has recently been suggested that the level of the endogenous glucocorticoid receptor stabilizing factor in mouse liver is regulated by the major hisiocompatibility, H-2. complex (Katsumata et al. [1]). We have developed an asssay for the activity of the endogenous heat-stable factor in mouse liver and have assayed this factor in liver cytosols prepared from two pairs of two H-2 congenic mouse strains. Our results show that the amount of the endogenous factor is the same in all four mouse strains and that it is not regulated by the H-2 locus.

In order to define the nature of the genetic lesion which gave rise to Sxr, the sex-limited autosomal dominant sex reversal condition in the mouse

We have investigated the changes occurring in the pattern of translatable mRNA species in germ cells during spermatogenesis of mice. Highly homogeneous cell populations of spermatocytes or spermatids were purified. Poly(A)+ mRNA was isolated from each cell population by oligo(dT)-cellulose chromatography. A comparison of meiotic and post-meiotic mRNAs was made by two-dimensional gel analyses of their in vitro synthesized translation products. Among spots identified in the fluorograms of two-dimensional gels, a number of qualitatively new proteins appeared after meiosis. The results suggest that some of poly(A)+ mRNAs are transcribed post-meiotically in haploid germ cells.