Robert P. Erickson

Post-meiotic gene expression

Evolutionary arguments and well-designed experiments (based on false premises, however) had suggested that post-meiotic gene expression did not occur in animals. The techniques of molecular genetics have now clearly demonstrated such genetic activity in mammalian testes. The current problem is to understand why some classes of genes, such as Zfy and many oncogenes, are expressed in this manner.

A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction

SummaryThe alphoid satellite family is the only repetitive DNA family showing chromosome specificity. We have developed a simple, rapid, and reliable test for sex diagnosis based on detection of these sequences in undigested genomic DNA using the polymerase chain reaction. In our test, dried blood specimens were the source of DNA. When female DNA was used as a template for the reaction, only the expected 130-bp X-chromosome-specific fragment was detected, while with male DNA both the expected 170-bp Y-chromosome-specific and X-chromosome-specific fragments were detected. The Y-chromosome-specific fragment was further characterized by restriction enzyme analysis. The Y fragment was detectable when DNA obtained from an equivalent of 10 μl of spotted blood was used in the reaction, whereas detection of the X fragment was possible with DNA from an equivalent of 5 μl of blood. Our test may find various applications in newborn screening and in forensic science.

Haploid accumulation and translational control of phosphoglycerate kinase-2 messenger RNA during mouse spermatogenesis

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.

Developmental program of PGK-1 and PGK-2 isozymes in spermatogenic cells of the mouse: specific activities and rates of synthesis.

Abstract 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 [ 3 H]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.

Is haploid gene expression possible for sperm antigens

The development of 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 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 makes 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 postmeiotic 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.

FUNCTIONAL ASSAYS FOR mRNA DETECT MANY NEW MESSAGES AFTER MALE MEIOSIS IN MICE

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.

H-2 and non-h-2 determined strain variation in palatal shelf and tongue adenosine 3':5' cyclic monophosphate. A possible role in the etiology of steroid-induced cleft palate.

The concentration of cAMP was measured in palatal shelves and tongues of 14.5‐day old foetuses, 14.5‐day old foetuses from steroid treated mothers, and 15.5‐day old foetuses from four inbred lines of mice which represent the four possible combinations of two H‐2 alleles and two residual genetic backgrounds. The incidence of spontaneous and steroid‐induced cleft palate in these four strains was also determined. Analyses of variance of the cAMP data reveal that both the H‐2 region and residual genetic background determine cAMP concentrations in both tissues and on both days of development. Similar analyses of cAMP concentrations after steroid treatments of the mother indicate that the interaction between H‐2 and residual genetic background is significantly different in the injected than in the uninjected mice in both palatal shelves and tongues. The incidence of steroid‐induced cleft palate parallels the palatal shelf concentration of cAMP before steroid treatment of the mother with one exception. These data suggest that a portion of the H‐2 controlled component of susceptibility to steroid‐induced cleft palate is mediated through alterations in the metabolism of cAMP.

Mannosidosis: Two brothers with different degrees of disease severity

Two siblings with different degrees of mental retardation, skeletal dysplasia, coarse facies, delayed speech, motor incoordination, recurrent respiratory infections, and immunological abnormalities, were found to have deficient alpha‐mannosidase activity. Cultured skin fibroblasts in one sib were markedly deficient in alpha‐mannosidase while all other lysosomal enzymes tested were within the normal range. The more severely affected sib came to autopsy and was found to have “washed‐out” appearing cortical neurons and marked histiocytosis effacing lymph node architecture and partially replacing the bone marrow. The post‐mortem brain and liver samples demonstrated a deficiency in alpha‐mannosidase relative to the elevations of other lysosomal enzymes. Although the patterns of abnormalities in the two cases closely match those of descriptions of “type II” and “type I” mannosidosis respectively, the variation should be due to genetic modifiers or environmental effects since the brothers must have shared similar alpha‐mannosidase mutations. Immunologic abnormalities present in the more severely affected sib suggest that the differential survival seen in mannosidosis types I and II may be due to differences in their immune systems.

Autosomal dominant polycystic liver disease: a second family.

An autosomal dominant pattern of transmission has been established for polycystic kidney disease. The degree of cystic involvement of other organs has been variable. The genetic pattern of transmission of polycystic liver disease independent of cystic kidney disease has never been established. We present a second family with polycystic liver disease without kidney disease. The lack of renal cysts is unlikely to be due to variable expressivity and penetrance of the gene for polycystic kidney disease. The liver cysts may be of late onset since none of the probands four children demonstrate cysts. Alternatively, none of these four individuals may have received the gene for polycystic liver disease from their affected mother. The family described supports an autosomal dominant pattern of inheritance for polycystic liver disease.

Familial occurrence of intracranial arterial occlusive disease (Moyamoya) in neurofibromatosis

Two out of six siblings with neurofibromatosis (in a sibship of eight) had clinical and roentgenographic evidence of Moyamoya‐type, intracranial arterial occlusive disease. This rare vascular complication of neurofibromatosis has not previously occurred among primary relatives. Several possible etiologies for such an association are discussed.