Ph.D. - Biomedical Sciences (Genetics - Cell, Molecular and Neuro Sciences)

Permanent URI for this collectionhttps://hdl.handle.net/10125/1934

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Applications of distribution theory in quantitative genetics
(University of Hawaii at Manoa, 1976) Yamashita, Toyoko S.
The present study was designed to develop procedures for estimating genetic parameters in a quantitative trait and detecting association between a quantitative trait and a genetic marker using a random sample of individuals from the population. An exact distribution of phenotypic values of a quantitative trait was used by which parameters such as number of loci involved, gene frequency at each locus, additive and dominant genotypic value, and the variance of environmental effects were estimated. When association between the quantitative trait and a genetic marker occurred, an additional parameter for the degree of linkage disequilibrium was introduced in the exact distribution. For testing the validity and usefulness of the procedures, individual phenotypic values were simulated by the computer with given parameters. The number of loci involved in the quantitative trait was 2, 5, and la, and gene frequency at each locus was chosen at random. Three heritability values were investigated; namely, 0.2, 0.5, and 0.8. The genotypic values were then calculated by setting degree of the dominance either to zero (no dominance) or one (complete dominance). The environmental effects were assumed to be normally distributed; however, the phenotypic distribution of the quantitative trait under investigation was not specified. Five replicate runs were made for most tests to allow variations in gene frequencies, each sample size being set to 5000. Smaller samples of 100 and 500 individuals were also tested. Based on the log likelihood function values, good estimations by the maximum likelihood method were obtained when heritability of the quantitative trait was moderately high (h^2 ≥.5). When the heritability value was low, the phenotypic distribution of the quantitative trait was mainly determined by environmental effects and, therefore, was expected to be normal by assumption. In this case, the method might not give consistent estimates of genetic parameters. As the number of loci increased, the phenotypic distribution would approach normal by the Central Limit Theorem; however, the procedure was still effective for traits with high heritability. Several cases for the association between one of the loci involved in the quantitative trait and a genetic marker were considered, including (1) complete independence (no linkage), (2) partial linkage, and (3) complete linkage or identical locus. The approach employed was mainly based on the distributions of phenotypic values. Data were subgrouped by the genotypes of the marker locus resulting in several phenotypic distributions, one for each genotype of the genetic marker. When association occurred between a quantitative trait locus and a genetic marker locus, the subclass distributions would be expected to be different from one another. The subsequent estimates of parameters including the degree of linkage disequilibrium were then derived from the exact distributions by the maximum likelihood method. The results were generally good. The present study using simulated data has clearly demonstrated that by employing the distribution of phenotypic values of the quantitative trait, it is possible to establish a genetic basis for the character from a random sample of individual data. When information on other genetic markers is available, the association between the quantitative trait and a genetic marker as measured by linkage disequilibrium may be estimated. However, further research would be needed to apply the procedures to real (non-simulated) data and to consider other parameters such as genotype-environmental interactions and epistatic effects in the phenotypic distribution.
Demonstration on zymograms and genetic studies of some enzymes from human saliva
(University of Hawaii at Manoa, 1975) Tan, Soon Guan
Saliva samples were collected, processed, concentrated and then subjected to polyacrylamide flat slab gel electrophoresis followed by staining to detect enzyme activities. Six different enzyme activities were detected out of the fifty enzyme staining procedures attempted. Repeatable and constant variants were observed in three of these, saliva acid phosphatases, esterases and glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase). Genetic studies were done on these three. Seven phenotypes were found for saliva acid phosphatases. Family and population studies suggested that these phenotypes are the products of two loci, Sap-A with three alleles, A, A' and O, and Sap-B with 1 three alleles, B, B^1 , and O. These loci were found to be polymorphic in Americans of Japanese and Caucasian ancestries living in the State of Hawaii. Saliva esterases show a complex picture on zymograms and had been divided into four major regions. Variants were observed in region 1, the fastest anodal migrating region. Three phenotypes had been observed in region 1. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles, Set-IF and Set-IS. This locus is polymorphic in Americans of Japanese and Caucasian ancestries. Region I esterases are carboxylesterases. Saliva glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) showed three phenotypes on acrylamide gel zymograms. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles, Sgd 1 and Sgd 2. All the above three saliva enzymes in which variants were observed thus allowing genetic studies to be done proved to be different from the analogous enzymes with similar functions in the red blood cell. They constitute previously undescribed polymorphisms in the human species. The three other enzymes whose presence in the saliva had been detected on zymograms were an oxidase, lactate dehydrogenase and superoxide dimutase. No variants were observed for these enzymes which would enable genetic studies to be done. Association and linkage studies were attempted between the three newly described polymorphic saliva enzymes among themselves and between them and the ABH secretor status, Lewis A substance in saliva, ABO blood group, C5, adenosine deaminase, esterase D, PGM I , haptoglobin, Gc protein, MN blood group, P blood group, Duffy blood group, Kell blood group and Kidd blood group loci using data from random Caucasian individuals. Significant associations were only found between Sap-A and haptoglobin locus and between Set-l and MN blood group locus. These significant associations are most probably due to chance. Linkage studies were not very fruitful because of the small numbers of informative families available due to the small family size in most of the sampled families. None of the accumulative lod scores yielded conclusive results.
Enzyme polymorphism and desiccation resistance in two species of Hawaiian Drosophila
(University of Hawaii at Manoa, 1974) Steiner, William W.M.
The genetics of pigeon populations on Oahu
(University of Hawaii at Manoa, 1972) Go, Rodney Chun-Pung
A genetic and epidemiological study of cleft lip and cleft palate in Hawaii
(University of Hawaii at Manoa, 1970) Ching, Gerald Hong Sung
The purpose of this study was (1) to obtain unbiased estimates of cleft incidences in the major racial groups in Hawaii, (2) to evaluate the extent and nature of racial differences in incidence, (3) to determine how the segregation of cleft defects varies among racial groups, and (4) to estimate the heritability of cleft defects using a multifactorial model of quasicontinuity. Cleft cases were ascertained through birth and death certificates and records of the Crippled Children Branch (Hawaii Health Department) and major Honolulu hospitals. The reference population which provided the basis for the analysis of incidence was composed of live births in Hawaii from 1948 to 1966, as compiled by Drs. Morton, Chung, and Mi. Live birth certificates provided demographic data on all individuals in the reference population. Variables included race and age of each parent, sex of child, birth order, year of birth, and occupation and military status of father. Families of probands were interviewed for additional data on types and numbers of affected and normal relatives and parental consanguinity. The ascertainment of CL(P) was estimated to be 97% complete, and that of CP to be 93% complete. The danger of relying exclusively on birth certificates for the ascertainment of cases was made clear by our findings that only 80.7% of CLP, 66.7% of CL, and 47.7% of CP probands born in 1948-1966 had their defects noted on their birth certificates. A number of factors were found to be related to the ascertainment probabilities of CP and CL(P). In the case of CP, ascertainment appeared more likely when the individual had three or more additional malformations and less likely when his father was actively engaged in military service. The case of CL(P) was more complicated. The probability of ascertainment of CL(P) apparently increased when the defect extended to the palate and when any additional malformation was present; it seemed to decrease with year of birth, age of father, death in infancy, and father in the military. Significantly, however, no racial biases in ascertainment were detected after adjustment for the effects of non-racial factors. After correcting for ascertainment biases, the estimated overall incidence of CP in Hawaii was .78 per thousand live births. In the pure Caucasian group, the estimated incidence was .50; in the pure Japanese group, it was .74. Incidences in excess of one per thousand were found in groups with Hawaiian ancestry. Regression analysis showed a definite association between Hawaiian parentage and high incidence of CP. This strong Hawaiian effect could not be attributed to maternal racial factors or to interracial hybridization. Orientals as a whole did not appear to have a significantly higher incidence of CP than Caucasians. For CL(P), the corrected incidence was estimated at 1.05 per thousand in Caucasians, 1.92 in Japanese, and 1.28 in the general population. Unlike in the case of CP, Hawaiian ancestry was not associated with high incidence of CL(P). Instead, the Oriental groups, particularly the Japanese and Filipinos (1.56), presented the highest risks. No maternal or hybridity racial effects were detected for any of the Oriental groups. The effects of non-racial factors (father's age, mother's age, birth order, year of birth, father's occupational and military status) on incidence were tested by separate regression analyses in the Japanese and in the Caucasian racial . groups. No consistent relationship between incidence and any non-racial variable was detected for either cleft type. However, CP incidence did appear to increase with lower occupational status in the Caucasian group, although not in the Japanese group. Estimates of mean segregation frequencies for CP were 2.2% in families of Hawaiian ancestry and 1.3% in all other families. Although the difference was not statistically significant, the higher risk in Hawaiian families would be expected under the multifactorial hypothesis which equates risk approximately to the square root of the population incidence. Heritability of CP was estimated to be 69%, which possibly indicates that additive genetic factors are very important in the etiology of CP. Mean segregation frequencies for CL(P) were 6.1% in pure Japanese, 6.4% in pure Filipinos, and 4.2% in all other groups. The higher risks in Japanese and Filipinos may reflect their higher incidences of CL(P). Overall heritability of CL(P) was estimated at 86%, with no significant differences among Japanese, Filipinos, and all other groups combined. The weight of evidence now seems to favor a multifactorial hypothesis of CL(P) inheritance, and the present estimates of heritability would suggest a large additive hereditary component.
Studies on quasi-continuity
(University of Hawaii at Manoa, 1969) Campbell, Mary Anne
This study was an attempt to fit a model of quasi-continuous variation to three sets of data. The model is based on the assumption that the trait under study is completely additive and wholly polygenic. Conditions due to a major gene, in the sense of classical Mendelian inheritance, would give unreasonable parameter estimates and a poor fit on such a model. Information on population prevalence and recurrence in sibs is used to estimate the parameters and predict risk for other degrees of relationship. Records on all surgically corrected cases of pyloric stenosis occurring in the period 1942 to 1966 in Hawaii provided the first set of data. Nearly 200 families were interviewed to obtain a pedigree and family history. Birth certificate numbers were matched with a file of all births for that time period to provide additional information on race and sociological variables. The sex ratio was similar to all earlier reports, approximately 4:1. The segregation analysis was compatible with a major gene hypothesis but the estimates had very large standard errors. The regression analysis of interracial crosses indicated a depression in the frequency in F1, indicative of recessivity. The fit to the model of quasi-continuity was neither good nor consistent. Such a theory cannot be eliminated as the possible genetic mechanism for pyloric stenosis, but the current model is inadequate to explain the data. Males with serum cholesterol levels greater than two standard deviations above the mean of a random sample of 7,000 forty to sixty year old Japanese males were selected for a study of hypercholesterolemia. Sibs were contacted, interviewed and serum cholesterol determinations made. The 219 sibships were analyzed by segregation analysis, providing an estimate of the segregation frequency of 0.52 and the proportion of sporadics as zero, evidence for a dominant gene. Estimates of heritability on the additive model exceeded 1.5. This condition appears to be a dominant gene with nearly complete penetrance. This finding is in agreement with other studies. Information from the Bureau of Identification provided a sample of individuals with a dermal ridge count of zero, an arch pattern on all ten fingers. These individuals were defined as "affected." Ridge counts were made for all available relatives. A control sample was selected from the Bureau files. The frequency of patterns and mean ridge counts for the Caucasian controls agreed well with published studies from England. The Japanese had higher mean counts and a significantly higher frequency of whorls. Correlation coefficients computed in the control sample for sib-sib and parent-child relationships agreed well with complete heritability. Segregation analysis in both the affected families and the controls failed to detect segregation of a major gene. Parameters were estimated for the quasi-continuous model using data from first degree relatives. Fitting these estimates to data from cousins and second degree relatives gave a non-significant deviation. Evidence from earlier studies has shown fairly conclusively that dermal ridge count is additive and multifactorial. The good fit of our data to this model of quasi-continuity was reassuring. Hypercholesterolemia, on the other hand, seems clearly due to a major gene. The failure to fit the data on pyloric stenosis may only reflect inefficient parameter estimates and lack of sufficient sophistication or accuracy in the current model.
A genetic study of the amylase isozyme polymorphism in Drosophila melanogaster
(University of Hawaii at Manoa, 1969) McCune, Thomas Brent
Many of the recently discovered genetically determined polymorphisms involving isozymes of Drosophila melanogaster have been concerned with identifying the enzyme biochemically and determining the formal genetics of the enzyme. After the survey of several enzyme systems in natural populations of Drosophila pseudoobscura by Lewontin and Hubby (1966), which indicated that isozyme polymorphisms are more frequent than previously expected, there has been greater interest in population studies of these isozymes as is shown by the recent work in Drosophila melanogaster concerning the Esterase 6 polymorphism (Yarbrough and Kojima, 1967) and the alcohol dehydrogenase polymorphism (Kojima and Tobari, 1969b). The purpose of this study was to investigate the frequencies of the amylase isozyme alleles in several natural populations of D. melanogaster, and on the success of this investigation, to attempt to see how the polymorphism might be maintained. The survey consisted of recently collected flies from natural populations of Texas and Wisconsin. In addition, the Odate population (collected in Japan four years ago and since kept in the laboratory) was also examined. For all surveys the frequency of the Amyl allele was clearly the most common (0.80) and was followed by Amy1,3 (about 0.12). Other less frequent alleles observed were Amy2,3, Amyl,2, and hmy1,6. Because of Amyl and Amy1,3 being consistently the most frequent in the natural populations, the selection studies concentrated on these two alleles in the Texas and Wisconsin populations. The results of the viability and fertility experiments (for matings within and between populations) consistently demonstrated higher viability and fertility for the Amyl,3/Amyl,3 genotype followed by Amyl/Amyl,3 and Amyl/Amyl genotypes respectively. The results of the developmental study, which employed the normal sugar containing media and a special media containing starch instead of sugar, were less consistent; however, the Amyl/Amyl genotype had the slowest developmental time with the Amyl/Amyl,3 and Amyl,3/Amyl,3 genotypes being similar to each other. From these results it is difficult to see why the Amyl has such a high gene frequency in natural populations. There was some indication from one of the viability experiments that the recovery frequency of the Amyl allele among progeny is higher than that of the Amyl,3 allele when the parents are very young, thus suggesting a prezygotic mechanism of some sort. This study, however, does not critically test for prezygotic selection and more specific tests are currently being planned.
Isolation and characterization of messenger ribonucleic acid for hemoglobin
(University of Hawaii at Manoa, 1969) Laycock, David Gerald
An 8s ribonucleic acid (RNA) species, isolated from rabbit reticulocytes, has been purified by salt fractionation and analyzed for characteristics of an information-carrying, messenger RNA. This RNA in reticulocytes is restricted to polyribosome complexes which are active in hemoglobin synthesis and has a base composition similar to that expected of a messenger RNA for rabbit globin. This RNA species has been assayed for the ability to support and direct protein synthesis in bacterial extracts. The 8s RNA from reticulocyte polyribosomes is translated when only N-acetyl-valyl- tRNA is present as an initiation residue. Both rabbit globin polypeptide chains have N-terminal valine residues. The protein produced in bacterial extracts directed by this RNA is identical to rabbit globin by chromatography on diethylaminoethyl-cellulose, carboxymethyl-Sephadex C50, paper electrophoresis and gel-filtration on Sephadex G75. The protein product combines with added hemin and the resultant complex is indistinguishable from native rabbit hemoglobin by the above criteria. The profile of tryptic peptides from chromatography on Dowex 50x2 indicates the product is N-acetyl globin. The implications of these results concerning the universality of the genetic code and of protein biosynthetic mechanisms at a macromolecular level are discussed.
Aberrant segregation in human populations
(University of Hawaii at Manoa, 1969) Grove, John Sinclair
Three cases of aberrant segregation in humans were considered. The first was a possible case of non-random assortment of chromosomes and meiotic drive. Separating children of fathers of given heterozygous ABO genotypes into sperm classes (classified by the ABO gene received from the father), for children of A1B and A2B fathers a change in association of the sperm type with the sex of the child (which would be determined by the father's sex chromosome received) occurred with advancing father's age. Specifically, the B sperm class was associated with the X chromosome when fathers were old and the Y chromosome when they were young. Mother's age and birth order effects were ruled out by multiple regression. This effect was strikingly non-linear: a fifth order polynomial of father's age was very highly significant (for pooled offspring of A1B and A2B fathers). The sex ratio changed within each sperm class in opposite directions. The segregation frequency of the B sperm class for female offspring increased with father's age (but not mother's age or birth order) and again a fifth order polynomial was very highly significant. The progeny of B0, A2O, and A1O plus A1A2 fathers were not affected. There was a non-linear change in segregation frequency of female offspring of AB mothers as mothers grew older which was not highly significant and may have been due to chance. The second study was on the genetic load of the population in Odate, Japan. Prenatal death was examined with respect to several factors and found to fall into at least three classes: early prenatal death (EPRE), up to four months of gestation; late prenatal death (LPRE), up to nine months; stillbirths, more than nine months. LPRE increased significantly (but non-linearly) with inbreeding but EPRE and stillbirths did not; regressions for LPRE and stillbirths differed significantly. Postnatal death was divided into three classes (which differed in several respects): early postnatal death (less than one month old); middle postnatal death (less than two years old); late postnatal death (two or more years old). Only early postnatal deaths increased significantly with inbreeding (.4 lethal equivalents per gamete; B/A = 14.5) and the regression differed significantly from those of the later stages. The last analysis was on ABO incompatibility in the same population. Although a preliminary analysis had not shown significant effects, it was found that the inclusion of three secretor interactions with ABO incompatibility showed very highly significant effects on the stages of mortality considered above. The secretor interactions were the mother's secretor type, the father's secretor type, and the probability that the zygote was See A and B incompatibility on 0 and non-O mothers were analyzed separately for mortality and were found to behave differently. Interactions with parity and (parity)^2 were often highly significant. The interactions tended to balance each other making simpler models non-significant. Some of the regressions suggested viability differences among genotypes of offspring. Significant environmental interactions were also found.
Erythrocyte isozymes, other polymorphisms, and the coefficient of kinship in northeastern Brazil
(University of Hawaii at Manoa, 1969) Azevêdo, Eliane S.
Application of zymogram development technique has become a powerful tool in genetics research. It provides easy detection of molecular variation in proteins, leads to the discovery of new polymorphisms, and significantly adds to the knowledgment of man's genetical makeup and population structure. This dissertation comprises a study of isozymes and other polymorphisms, followed by their application to the analysis of population structure. During the year 1962-63 a total of 1068 families was selected from a migrant population of northeastern Brazil. Red blood cells and sera were collected and have been kept in satisfactory condition for study of enzymes and proteins. The red cell enzymes glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, phosphoglucomutase and adenylate kinase were studied by starch gel electrophoresis. Except for G6PD whose gene frequencies depend on the proportion of Negro ancestry, no other association was detected when gene frequency was regressed on other variables such as social level, sex, age, longitude, latitude, and regional pathology (hepatosplenomegaly). Two families carrying an unusual variant in the G6PD system are described, accompanied by biochemical characterization of the new allele v and cross-check with other G6PD mutants. Once proved to be unique, the new all8le is provisionally designated as Gd Minas Gerais (MG), after the Brazilian state of the propositus. Three families carrying rare alleles at the PGMZ locus are also described. A linkage study between isozymes and other genetic markers was done, mainly stimulated by a recent report claiming a probable linkage between the phenylthiocarbamide (PTC) and the phosphoglucomutase (PGM1 ) loci. We studied twenty-eight families where one of the parents was doubly heterozygous, i.e., double and single backcrosses, and four intercross families. We did not detect linkage in our sample either by analyzing the recombination frequencies by sex, nor in the pooled sample. Most lod scores are negative, giving evidence against linkage, and close linkage (θ ≤ .2) may confidently be excluded. By using other segregating families we also tested linkage between hemoglobin and PGM, hemoglobin and Gc, hemoglobin and cholinesterases E1 and E2. None of the observed lod scores were suggestive of linkage. The isozyme, and other polymorphisms which were previously studied in the same sample, are used to bioassay the population structure of northeastern Brazil. The migration model of Malecot was selected because it estimates the most pertinent parameters ruling the structure of a population (migration, systematic pressure, and consanguinity) besides its many-sided applicability to any genetic system (phenotype), isonomy, metric data, and pedigree inbreeding. A computer program DISTAN draws random pairs of individuals, groups them into classes according to the distance between their birth-places, and estimates the coefficient of kinship for every distance class. Estimation of the parameters a (mean coefficient of kinship for individuals born at small distance), b (measure of systematic pressure), and £ (dimensionality of migration) are also given by the program DISTAN. According to Malecot's theory, if the distance between birth-place of individuals I-and J is d, then the coefficient of kinship is given by φ(d) = ae^-bd d^-c , and is defined as the probability that two alleles drawn at random, one from individual I and one from individual J, be identical by descendent from a common ancestor. The monotonic decrease with distance of inbreeding and kinship predicted by Malecot has been observed in northeastern Brazil for polymorphisms, metrics, surname concordance (isonomy), and pedigree inbreeding. Comparison of these indicators with each other and with Malecot's theory indicates that consanguineous marriage at a given distance is preferential, most conspicuously at large distances. The mean coefficient of inbreeding is estimated to be .0080, of which 70 per cent was ascertained through pedigrees. Interpretation of these results is discussed in terms of various concepts of population structure.
Non-random assortment of chromosome pairs and meiotic drive in Drosophila melanogaster
(University of Hawaii at Manoa, 1968) Sakai, Richard Kazuichi
During meiosis different chromosome pairs are thought to assort quite randomly from each other; thus, an A/a; B/b (A and B refer to different chromosome pairs) chromosome type would be expected to produce four kinds of gemetes, AB, Ab, aB and ab in equal numbers. Several papers have been published reporting that in some cases the above principle was not always true (Novitski and Sandler, 1957; Grell, 1959). In all of these studies, however, the genotypes used were characterized by abnormal chromosome complements or involved chromosomes of more or less abnormal structure such as trans locations or inversions. In the absence of such abnormalities it has been widely assumed that the principle of random assortment is generally true. Recently, however, Hiraizumi and Nakazima (1967) reported that when the second chromosome of £. melanogaster carried segregation distorter but was free of any visible structural abnormality, it tended to assort more frequently with the Y chromosome. They also observed that the amount of non-random assortment was positively correlated with the frequency of recovery of the SD-bearing chromosome. The above phenomenon was well established for the case of the SD system but it is possibly a unique phenomenon characteristic only of this system. If, however, the phenomenon is generally true in nature, then it may be one of the important factors changing gene frequencies in populations. The present investigation established the generality of non-random assortment between the second and sex chromosomes and confirmed the positive correlation between the segregation frequency of the second chromosome and the degree of non-random assortment. Preliminary mapping of the elements of the system was also done. These findings are of interest because in many Drosophila experiments fitnesses of genotypes have been estimated by differential larval stage viabilities based upon the recovery frequencies among progeny flies under the assumption that the distorted segregation was due solely to the differential viabilities of the genotypes involved. with the establishment of the generality of the phenomenon of non-random assortment of chromosome pairs and the changes in segregation frequencies associated with it, many of these conclusions may have to be critically re-examined.
The genetical structure of northeastern Brazil
(University of Hawaii at Manoa, 1966) Yasuda, Norikazu
Inbreeding effects in northeastern Brazil
(University of Hawaii at Manoa, 1966) Krieger, Henrique
The present study deals with the analysis of the inbreeding effects on a migrant Brazilian population and comparison with other studies. This population (comprising 1068 families with more than 9000 pregnancies) was found to have a good homogeneity between the inbred and control groups with respect to several socio-economic and biological variables. Inbreeding did not have a measurable effect on body weight, height, or hematocrit. These results are compatible with a small dominant component in the inheritance of the traits in question. However, diastolic blood pressure showed a significant increase with inbreeding. Diastolic blood pressure increased by 3.6 mm Hg per 10% of inbreeding. Although this association could well be spurious, the need for further investigation clearly emerges, not only in connection with the present results, but also with the results of Nance and collaborators of an apparent association of diastolic blood pressure and the ABO blood group system. Turning to mortality, there was a complete lack of inbreeding response of both stillbirths and postnatal deaths; however, abortions plus miscarriages showed a significant inbreeding effect (of both the mother and fetus). Our interpretation is that two opposing forces are being measured. On one hand recessive lethal genes tend to increase the risk of inbred fetuses and on the other hand genes involved in incompatibility mechanisms lead to a smaller death risk. This hypothesis is based on the observed interaction effects of inbreeding of the mother and inbreeding of the zygote, with parity. In an attempt to study the inbreeding effect on early and undetected abortions, we used the fraction of the cohabitation time during which the mother was not known to be pregnant or in postpartum infertility as a parameter that would measure a sterile period due to embryonic loss if all the other concomitant variables are randomly distributed with respect to F. "Waiting time" for a mother to become pregnant was distributed independently of F, thus providing no evidence for a large number of lethal equivalents acting at this period of life. The best approach available today to resolve segregation and mutation loads is the use of the double binomial model of segregation analysis. This was applied for the malformation and early fetal death data in the present material and showed that sporadic cases are not associated with inbreeding, which is inconsistent with the "phenodeviant" hypothesis. Also the present data did not show any heterosis in interracial crosses, as claimed by some proponents of phenodeviants. Comparison of estimates from different populations suggests that non-genetic mechanisms are confounded with effects of inbreeding and interracial crosses in some (perhaps many) studies. The B/A ratio of inbreeding effect to panmictic load has little power to isolate the mutational load unless supplemented by both reliability tests and segregation analysis. When the latter method reveals that inbreeding effects are limited to high-risk families, as in our material, this provides strong evidence for a mutational load.
Genetic isozyme variations in Zea mays
(University of Hawaii at Manoa, 1965) Scandalios, John G.
In an effort to narrow the gap in knowledge between the primary action of the gene and" the phenotype (in maize), use has been made of the technique of starch gel electrophoresis in combination with various enzyme staining procedures. The first to apply these methods to plant biochemical genetic studies was Schwartz (1960), who described genetic variations in esterases in Zea mays. In the present thesis slightly modified techniques were used to describe variations in the enzymes leucine aminopeptidase (LAP), catalase, esterase, peroxidase and amylase, in maize. Four different Molecular forms (isozymes) of LAP were found in maize endosperm (zones A, B, C, and D). The LAP A and LAP D enzymes show electrophoretic variations, and each was found to be controlled by a pair of alleles acting without dominance. Linkage between the two loci has been demonstrated. Tissue studies showed that the A and B zones were distributed throughout the organism, while the C zone was common only to the embryo and endosperm. The 0 zone was found only in the endosperm. The presence of tissue-specific variants of LAP and also of esterase, peroxidase and catalase, as demonstrated in 'these studies, lend supporting evidence for the existence of multiple molecular forms of various enzymes within" the same organism and within the same tissue; a point of great significance in developmental biology. The polymorphism found in catalase is of special interest due to the formation of hybrid enzymes in the heterozygote, which have electrophoretic mobilities intermediate to those of the parental types. The number and the activity concentration of these hybrid enzymes suggested that maize catalases exist as tetramers and that the hybrid patterns could be the result of random combination of two different catalase subunits. That catalase exists functionally as a tetramer is supported by experiments in which mixtures of two variants were frozen in NaCl and were dissociated into the "presumed" monomers. On thawing, reassociation into functional tetramers occurred. Such findings suggest that the primary product of catalase genes may be the monomer and that the enzymatically active unit is an aggregate (tetramer) of such monomers. The amylases are also of special interest developmentally, since their activity appears to be confined to the early period of germination and at a time when starch degradation is expected. Preliminary immunochemical studies are suggestive of differences in antigenic specificity of the variants mentioned. The possible significance of these findings in reference to gene action, development, and evolution of isozymes is discussed.
A genetic study of the RA cell, a new line of human amnion, with special reference to cytogenetics, radiation effects and enzyme systems
(University of Hawaii at Manoa, 1964) Regan, James Dale
When working with a continuous line of human cells, it is particularly useful to be able to secure an adequate supply of normal primary tissue of the same kind. Thus genetic characteristics such as chromosome constitution and the presence or absence of certain enzymes can be compared in the primary and continuous cells. With such epithelioid lines as Chang liver (Chang, 1954) or the several lines derived from human marrow by sternal puncture (Szybalski, 1963), the securing of comparative primary tissue is troublesome. By contrast human amnion cells are easy to obtain in large quantities from normal placentae. The number of continuous lines of spontaneously altered human amnion cells that have been reported in the literature is surprisingly small. Only five are listed by Hayflick and Moorhead (1962) and only three have appeared with any frequency; the FL line (Fogh and Lund, 1957) the WISH amnion (Hayflick, 1961) and the Fernandez line (Fernandez, 1958). Not all of these are equally useful, e.g., one investigator (Loh, personal communication) has found the FL cell to be somewhat poor in cultural properties for virological studies. It is therefore believed that continuous lines of spontaneously altered human amnion cells with favorable growth characteristics and relatively minimal fastidious care can be extremely useful in studies of human genetics in vitro, and in other areas of inquiry. The purpose of this report is to describe in detail such a line of cells, the RA cell. A brief description could be given in a paragraph, relating the origin of the cells and stating the medium required for their cultivation. In fact, a considerable number of cell lines have appeared in the literature with little or no description. This occurrence seriously limits their usefulness, however. In this regard it is of interest that some recent studies of some well-known cell lines, using immunological and cytogenetic methods have yielded rather surprising results. Brand and Syverton (1962) found that 17 lines of cells supposedly derived from human, monkey, rabbit, swine, calf, hamster and duck tissues did in no case belong to the supposed species of origin but were either mouse or human cells. Several otherwise excellent genetic studies have appeared where the cell being used could simply no longer be identified (Lieberman and Ove, 1959, 1959a). It is believed, therefore, that a more extensive description of the RA cell is justified. In addition to the merely descriptive material, experimental studies with the RA cell reported here indicate the cell can be very useful for several aspects of cell culture genetics, including cytogenetic, x-ray resistance and enzyme studies.
Artificial induction of polyploidy in orchids by the use of colchicine
(University of Hawaii at Manoa, 1960) Nakasone, Henry Y.
The study of chromosome numbers of p1.ants has shown the occurrence of a large number of natural polyploids. In :related species of plants entire series of polyploids have been revealed. Muntzing (1) indicates that at least 50 per cent and probably more of the angiosperms are polyploids and that they have played an important role in the evolution of plant species. Presumably, polyploids were able to invade new habitats because of unusual vigor, growth, and general adaptability. Plant breeders and geneticists have become aware of the increased fertility of the naturally occurring amphidiploids over their ancestral hybrids. Cytological investigations have demonstrated that many plants selected from the wild for domestication are polyploids. These were selected because of specific, superior values even though the ploidy of these plants were unknown at that time. With increased knowledge of the nature of polyploids and the realization of their importance in plant improvement programs and in genetic studies, breeders have specifically bred for polyploid types. They have concluded that in many instances polyploids are superior to their diploid counterparts in vigor, size, quality, texture, and other characteristics. In the orchid industry, polyploidy has come to assume a prominent role in the production of superior types. Kamemoto (32) has shown that the award winning Cattleya hybrids such as C. Balmar, C. Bow Bells, and C. Joyce Hannington were triploids. Storey (57, 58) and. Kamemoto (33, 34) showed that superior selections of hybrids in the vandaceous group were triploids, tetraploids, and pentaploids. Kosaki (36) working with Dendrobiums, showed that a number of superior plants were triploids and tetraploids. Polyploid plants developed in the early years of orchid breeding originated through a number of avenues. Many occurred through spontaneous doubling in the somatic tissues and non-reduction of gametes, while others were the result of polyspermy and polyploidization of gametes. Many present day advance generation hybrids are the results of hybridization of these polyploids. In the course of an extensive breeding program in orchids, relatively high sterility among intergeneric hybrids and to a lesser degree among interspecific hybrids was encountered. The cytological basis for such sterility in orchids is largely that of non-homology of the chromosomes (32, 57, 58, 33, 34). Because of the highly sterile nature of many diploid hybrids as well as triploid strains, it was found desirable to overcome this sterility barrier in order to advance the breeding program. Previous workers have demonstrated with plants other than orchids that this sterility barrier could be removed by doubling the chromosome numbers of the sterile types, and that certain chemicals such as colchicine are effective in inducing such changes. Thus far, attempts that have been made both here and in other areas indicate considerable difficulty in inducing polyploidy artificially in orchids. Furthermore, no studies have been conducted on the behavior of such induced polyploids in orchids, and very little work has been done relative to comparison of morphological differences between diploids and induced polyploids. Following upon the above considerations, the thesis as presented here involved three major objectives. The first objective was to devise practical methods for inducing polyploids in selected orchid groups by the use of colchicine. The second objective entailed the study of the characteristics of the induced polyploids, with these types being compared with their diploid counterparts in respect to morphological differences. The third objective involved an attempt to analyze and explain the induction of cytochimeras found among most of the induced polyploids.
Paragenetic studies of TgHbox1 during sea urchin embryogenesis
(University of Hawaii at Manoa, 1995) Turano, Brian
The HOM-C homeobox genes are responsible for regional specification along the anterior-posterior axis of the embryo which results in the determination of body structures. TgHbox1, an Antennapedia-class homeobox gene related to homeobox genes in the HOM-C cluster has been previously described in the sea urchin Tripneustes gratilla (Dolecki et aI., 1986). This dissertation is an investigation into the function of the TgHbox1 gene during sea urchin embryogenesis. Due to the lack of the ability to perform classical genetic studies to examine gene function in the sea urchin, a paragenetic approach was designed. This was accomplished by microinjection of batches of sea urchin eggs with one of the following gene-specific reagents: phosphorothioate-modified oligonucleotides with TgHbox1 antisense sequence, TgHbox1-specific polyclonal antibodies, a synthetic lacZ-TgHbox1 fusion mRNA encoding a potential dominant negative protein, or a bacterially expressed truncated TgHbox1 protein to inhibit function of the endogenous protein. The embryos were fertilized and followed visually through embryogenesis. The authenticity of all reagents was confirmed in vitro as well as in vivo. Additionally, the persistence of the reagents or their products were demonstrated to be in excess of the endogenous target molecules and to be present in the embryo during the relevant development stages. Morphological analysis of the embryos injected with the four different TgHbox1-specific reagents yielded no specific mutant embryonic phenotype or perturbation of embryogenesis. The findings suggest are consistent with any of the following: TgHbox1 function was not inhibited sufficiently by any of the gene-specific reagents, redundant homeobox genes or other genes were able to functionally compensate for the loss of TgHbox1 gene function, TgHbox1 has no function in the stages of development studied, or there was an unidentified conceptual or experimental flaw in each of the four approaches.
pp60src-mediated phosphorylation of connexin43, a gap junction protein
(University of Hawaii at Manoa, 1995) Loo, Lenora Weing Moun
Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease In gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Confocal microscopy data indicates that the two proteins are within physical proximity allowing for a potential kinase-substrate interaction. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43 we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially-purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60src. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. This phosphorylation event occurred in the COOH-tail region of the Cx43 molecule because pp60src phosphorylated a GST-fusion protein containing the COOH-tail region of Cx43 and the phosphotryptic peptides generated using this construct comigrated with the subset mentioned above. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60v-src and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this dissertation demonstrates that kinase active pp60src is capable of phosphorylating Cx43 In a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down regulation of gap-junctional communication, these results suggest that pp60v-src regulates gap junctional gating activity via tyrosine phosphorylation of Cx43.
Mechanistic studies of carotenoid cancer chemopreventive action in mammalian cell cultures: involvement of gap junctional communication and connexin43 gene expression
(University of Hawaii at Manoa, 1992) Zhang, Li-Xin

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