Human transferrin polymorphism

The application of isoelectric focussing (IEF) has revealed a large amount of heterogeneity in the human transferrin (TF) system and has enhanced its potential value in anthropological and genetic studies. The average heterozygosity has been elevated from 0.05, observed by conventional methods of electrophoresis, to 0.29 detected by IEF. So far approximately 30,000 individuals from 122 population groups have been analyzed for TF subtypes to evaluate the magnitude of genetic variation at the TF locus. Possible environmental and biological factors, which may be operating to maintain the TF polymorphism, are discussed.     

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.     

DNA “fingerprints” and paternity ascertainment in chimpanzees (Pan troglodytes)

Highly variable regions of DNA are found in a wide diversity of organisms and are typically composed of alleles consisting of a variable number of tandem repeats (VNTRs) of a short core sequence. DNA fingerprinting probes are VNTR probes that simultaneously detect a large number of similar VNTRs in the target DNA. The highly polymorphic pattern observed in a DNA fingerprint allows resolution of questions concerning individual identification. M13 phage was used to fingerprint captive chimpanzees for paternity ascertainment. Although the probability of band sharing among captive chimps appears to be higher than among some other reported captive and feral animal populations, the probe is highly useful and can be expected to become more widely used in the genetic management of captive populations.     

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp60c-src, primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60c-src function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets.     

Genetic studies of human apolipoproteins. V. A novel rapid procedure to screen apolipoprotein E polymorphism

A simple and new method has been developed to detect apolipoprotein E polymorphism directly from plasma or serum without prior ultracentrifugation and delipidation. The method combines the use of dialyzed plasma or serum samples with or without neuraminidase treatment followed by monodimensional isoelectric focusing in simple or 3 M urea gels at a constant low power and progressively increasing voltage over a 3-hr period, and finally protein blotting to a nitrocellulose membrane. Apolipoprotein E phenotypes are identified immunologically using a double antibody reaction, the primary antibody being a monospecific, polyclonal goat anti-apolipoprotein E, and the secondary antibody being a rabbit anti-goat IgG conjugated with alkaline phosphatase. The method was employed to screen apolipoprotein E polymorphism in two white populations in the United States. The frequency values are comparable to those reported previously by other investigators using conventional detection methods. The procedure is simple, accurate, suitable for large scale epidemiologic, clinical, and genetic studies.     

Comparison of five tandem repeat loci between humans and chimpanzees.

Five tandem repeat loci were studied in humans and chimpanzees using VNTR probes derived from human DNA. Shared alleles were found at three loci and were often the modal allele in one species but never in both. There was no difference in the mean number of alleles per locus. However, these species exhibited substantially different levels of gene diversity, with chimpanzees monomorphic at two loci. Evidence of reduced variability in chimpanzees corroborates earlier comparisons using isozymes and plasma proteins. Molecular mechanisms, population dynamics, or both may be responsible for these differences. Equal numbers of alleles per locus may reflect high mutation rates. By one test, chimpanzees were out of equilibrium at one locus, which may reflect a typing error or population substructure. The long divergence time, and the high probability of backward mutations, precludes accurate estimation of genetic distance between these species.     

Separation of phosphoinositides and other phospholipids by two-dimensional thin-layer chromatography

A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.     

Individual admixture estimates: disease associations and individual risk of diabetes and gallbladder disease among Mexican-Americans in Starr County, Texas

The ethnic and geographic distributions of several common chronic diseases show distinct patterns that are consistent with the distribution of genes and genetic admixture. For example, diabetes and gallbladder disease occur most frequently among Amerindians, while those genetically admixed with them (such as Mexican-Americans) have intermediate rates, and lowest rates are found among Whites and Blacks. Because there will be heterogeneity from individual to individual in ancestral affinity within an admixed population, a method is developed for estimating each person's admixture probability. Results confirm that there is substantial heterogeneity of individual admixture among Mexican-Americans in Starr County, Texas, with a mean value indicating that 65% of genes in this population are Caucasian derived and 35% Amerindian derived. The individual estimates are shown to be unrelated to the probability of being diabetic and only marginally related to gallbladder disease, with those having the most Amerindian affinity being at increased risk. These results are a consequence of the independent assortment of loci and indicate that unless the markers employed are related (including linkage) to the disease of interest, the method will have limited utility. Individual admixture estimates will be useful, however, for examining aspects of population structure and will find increased utility for predicting disease and examining disease associations as more and more of the genome is represented by markers, a very probable prospect with the abundance of DNA polymorphism being identified by restriction enzymes.     

Genetic studies of low abundance human plasma proteins. II. Population genetics of coagulation factor XIIIB.

Plasma samples from a large number of different ethnic groups, consisting of U.S. whites, U.S. blacks, Eskimos from Kodiak and St. Lawrence Island, Aleuts of the Pribilof Islands, and three Amerindian groups from Canada and Mexico have been analyzed by isoelectric focusing followed by immunoblotting to determine the magnitude of genetic variation at the F XIIIB structural locus. The synthesis of published data and our new data demonstrate remarkable variation in the distribution of the three common alleles at this locus and establishes F XIIIB as an extremely informative marker for population differentiation and evolutionary studies. Genetic distance analysis based on this variation separates Caucasian, black, and Mongoloid populations into three distinct clusters.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.