Studies of the Inheritance of Human Ribosomal DNA Variants Detected in Two-Dimensional Separations of Genomic Restriction Fragments

We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separations of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in ~20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.     

Lck-dependent tyrosyl phosphorylation of the phosphotyrosine phosphatase SH-PTP1 in murine T cells.

The phosphorylation and dephosphorylation of proteins on tyrosyl residues are key regulatory mechanisms in T-cell signal transduction and are controlled by the opposing activities of protein tyrosine kinases and phosphotyrosyl phosphatases (PTPs). In T cells, several nontransmembrane protein tyrosine kinases are associated with receptors; for example, Lck is bound to the coreceptors CD4 and CD8 and becomes activated upon their stimulation. In comparison, little is known about the role of nontransmembrane PTPs in early T-cell signaling. SH-PTP1 (PTP1C, HCP, SHP) is a nontransmembrane PTP expressed primarily in hematopoietic cells, including T cells. We have found that SH-PTP1 is basally phosphorylated on serine in resting T cells. Upon stimulation of CD4 or CD8 either in a T-cell hybridoma cell line or in primary thymocytes, SH-PTP1 becomes tyrosyl phosphorylated. Moreover, SH-PTP1 is constitutively phosphorylated on tyrosine in the Lck-overexpressing lymphoma cell line LSTRA. SH-PTP1 is also a good substrate for recombinant Lck in vitro. Comparisons of the tryptic phosphopeptide maps of wild-type SH-PTP1 and deletion and point mutations establish that the two sites (Y-536 and Y-564) which are directly phosphorylated by Lck in vitro are also phosphorylated in vivo in LSTRA cells. One of these sites (Y-564) is phosphorylated in T cells in response to Lck activation. We conclude that SH-PTP1 undergoes Lck-dependent tyrosyl phosphorylation in T cells and likely plays a role in early T-cell signaling.     

Alterations in G-proteins in congestive heart failure in cardiomyopathic (UM-X7.1) hamsters

In order to explain the attenuated sympathetic support during the development of heart failure, the status of -adrenergic mechanisms in the failing myocardium was assessed by employing cardiomyopathic hamsters (155–170 days old) at moderate degree of congestive heart failure. The norepinephrine turnover rate was increased but the norepinephrine content was decreased in cardiomyopathic hearts. The number and the affinity of receptors in the sarcolemmal preparations were not changed in these hearts at moderate stage of congestive heart failure. While the basal adenylyl cyclase activity was not altered in sarcolemma, the stimulation of enzyme activity by NaF, forskolin, Gpp(NH)p or epinephrine was depressed in hearts from these cardiomyopathic hamsters. Since G-proteins are involved in modifying the adenylyl cyclase activity, the functional and bioactivities as well as contents of both Gs and Gi proteins were determined in the cardiomyopathic heart sarcolemma. The functional stimulation of adenylyl cyclase by cholera toxin, which activates Gs proteins, was markedly depressed whereas that by Pertussis toxin, which inhibits Gi proteins, was markedly augmented in cardiomyopathic hearts. The cholera toxin and pertussis toxin catalyzed ADP-ribosylation was increased by 37 and 126%, respectively; this indicated increased bioactivities of both Gs and Gi proteins in experimental preparations. The immunoblot analysis suggested 74 and 124% increase in Gs and Gi contents in failing hearts, respectively. These results suggest that depressed adenylyl cyclase activation in cardiomyopathic hamsters may not only be due to increased content and bioactivity of Gi proteins but the functional uncoupling of Gs proteins from the adenylyl cyclase enzyme may also be involved at this stage of heart failure.     

A "disproportion" between the frequency of rare electropmorphs and enzyme deficiency variants in Amerindians

Our previous studies have revealed a higher frequency of nonpolymorphic electrophoretic variants in blood samples from Amerindians than in similar samples from Caucasians and Japanese. Our present study finds, by contrast, that the frequency of deficiency variants of 11 erythrocyte enzymes, sampled in nine Amerindian tribes of Central and South America, is essentially the same (1.5/1,000 determinations) as in Caucasians or Japanese. Possible explanations of the elevated frequency of mobility variants in the tropical-zone/ unacculturated populations include: higher mutation rates resulting in both electrophoretic and activity variants in Amerindians but increased selection against deficiency variants in the Amerindians, or comparable mutation rates in both populations coupled with a greater probability of a mobility variant attaining a relatively high frequency among the Amerindians.     

Two more "private" polymorphisms of Amerindian tribes: LDHb GUA-1 and ACP1 B GUA-1 in the Guaymi in Panama.

A survey of Guaymi Indians of Panama for the occurrence of genetic variants of 25 proteins of the erythrocytes and sera have revealed, in addition to seven well-known genetic polymorphisms, four rare variants and two "private polymorphisms," the latter involving erythrocyte acid phosphatase and lactate dehydrogenase. The significance of such private polymorphisms in tribal populations to the interpretation of rare variants in civilized populations is emphasized.     

Genetic studies of the Macushi and Wapishana Indians

Summary Blood samples from 509 Macushi and 623 Wapishana Amerindians of Northern Brazil and Southern Guyana have been analyzed with reference to the occurrence of rare variants and genetic polymorphisms of the following 25 systems: (i) Erythrocyte enzymes: acid phosphatase-1, adenosine deaminase, adenylate kinase-k, carbonic anhydrase-1, carbonic anhydrase-2, esterase A_1,2,3, esterase D, galactose-1-phosphate uridyltransferase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, nucleoside phosphorylase, peptidase A, peptidase B, phosphoglucomutase 1, phosphoglucomutase 2, phosphogluconate dehydrogenase, phosphohexoseisomerase, triosephosphate isomerase and (ii) Serum proteins: albumin, ceruloplasmin, haptoglobin, hemoglobin A, hemoglobin A₂ and transferrin. Fifteen different rare variants were detected, involving 11 of these systems. In addition, a previously undescribed variant of ESA_1,2,3 which achieves polymorphic proportions in both these tribes is described. Excluding this variant, the frequency of rare variants is 1.1/1000 in 12510 determinations in the Macushi and 4.7/1000 in 15 396 determinations in the Wapishana. The ESA_1,2,3, polymorphism was not observed in 382 Makiritare, 232 Yanomama, 146 Piaroa, 404 Cayapo, 190 Kraho and 112 Moro. Irregularities in the intratribal distribution of this polymorphism in the Macushi and Wapishana render a decision as to the tribe of origin impossible at present. Gene frequencies are also given for previosly described polymorphisms of 5 systems: haptoglobin, phosphoglucomutase 1, erythrocyte acid phosphatase, esterase D, and galactose-1-phosphate-uridyl-transferase.Research supported by the National Science Foundation and the Energy Research and Development Administration.     

Avian erythroblastosis virus produces two mRNA''s.

We analyzed the viral mRNA's present in fibroblast nonproducer clones transformed by avian erythroblastosis virus. Two size classes of mRNA (28 to 30S and 22 to 24S) were identified by solution hybridization with both complementary DNA strong stop and complementary DNA made against the unique sequences of avian erythroblastosis virus. Based upon the kinetics of hybridization with complementary DNA made against the unique sequences of avian erythroblastosis virus, we estimated that there were 400 to 500 copies of the 28 to 30S RNA per cell and 200 to 250 copies of the 22 to 24S RNA per cell. Both RNA species were packaged in the virion. In vitro translation of the 28 to 30S virion RNA yielded a 75,000-dalton protein which was the 75,000-dalton gag-related polyprotein found in avian erythroblastosis virus-transformed cells. In vitro translation of the 22 to 24S virion RNA yielded two proteins (46,000 and 48,000 daltons). This indicates that there may be two genes in avian erythroblastosis virus, one coding for the 75,000-dalton gag-related polyprotein and the second coding for the 46,000- or 48,000-dalton protein or both.