Technique Of Starch-gel Electrophoresis Research Articles

GENETIC DIFFERENTIATION IN THE FLORIDA SUBSPECIES OF HELIANTHUS DEBILIS (ASTERACEAE)

Techniques of horizontal starch gel electrophoresis were employed to estimate levels of genetic variation within and genetic differentiation among populations of the three Florida subspecies of Helianthus debilis. The subspecies are H. d. debilis, H. d. vestitus, and H. d. tardiflorus. These taxa are very similar with respect to levels of genetic variability. The average values across all populations for proportion of polymorphic loci (25.5%) and number of alleles per polymorphic locus (2.35) are comparable to those found in other outcrossing plants. The average frequency of heterozygotes per locus (0.05) is lower than that found in other outcrossers. Local populations within each subspecies are genetically very similar. The average genetic distance between local populations is D = 0.010 ± 0.001. Subspecies vestitus and subspecies tardiflorus are also genetically very similar (D = 0.015 ± 0.001). However, subspecies debilis is differentiated from both subspecies vestitus (D = 0.121 ± 0.006) and subspecies tardiflorus (D = 0.103 ± 0.005). This genetic differentiation parallels morphological differentiation. The average genetic distance among all three subspecies is D = 0.080 ± 0.003. A moderate amount of genetic differentiation accompanies the process of subspeciation in Helianthus debilis.

Population genetics of phosphoglycolate phosphatase

A total of more than 3700 individuals from 31 different populations throughout the world have been phenotyped for the red cell enzyme phosphoglycolatc phosphatase using the technique of starch gel electrophoresis and the frequencies for variant alleles have been determined. The Australian Aboriginal and Papua New Guinean populations tested were invariant as were most populations from southeast Asia and a number of Pacific Islands. White Australian, Indian, Lebanese, Iranian and Armenian groups were all polymorphic for PGP2 and some also for PGP3. The most notable finding was in the Amerindian populations where the frequency of PGP2 was more than 30% for some groups. A total of more than 3700 individuals from 31 different populations throughout the world have been phenotyped for the red cell enzyme phosphoglycolatc phosphatase using the technique of starch gel electrophoresis and the frequencies for variant alleles have been determined. The Australian Aboriginal and Papua New Guinean populations tested were invariant as were most populations from southeast Asia and a number of Pacific Islands. White Australian, Indian, Lebanese, Iranian and Armenian groups were all polymorphic for PGP2 and some also for PGP3. The most notable finding was in the Amerindian populations where the frequency of PGP2 was more than 30% for some groups.

Evolutionary Genetics of Birds II. Conservative Protein Evolution in North American Sparrows and Relatives

Avise,J. C.,J. C. Patton, and C. F. Aquadro (Departments of Zoology and Genetics, University of Georgia, Athens, Georgia 30602) 1980. Evolutionary genetics of birds II. Conservative protein evolution in North American sparrows and relatives. Syst. Zool., 29:323-334.-Differentiation at 20-21 protein-coding genes was examined by conventional techniques of starch-gel electrophoresis among twelve species and seven genera of North American sparrows and relatives, Emberizidae, subfamily Emberizinae. One species representing Fringillidae was also included. Data were summarized in a distance matrix which was subsequently used to infer phylogenetic trees by a variety of methods. Results were generally consistent with current classification. Two salient results were unanticipated: 1) the relatively close genetic similarity of Pipilo to group I Emberizinae; 2) the relatively large genetic distance of Calcarius from other Emberizinae. A search of the literature revealed that the distribution of a behavioral characteristic, bilateral scratching, had led to a prediction of phylogenetic relationships for these genera fully consistent with the protein information. This result is significant because it lends support to proposals that some behavioral traits are extremely valuable phylogenetic markers. Levels of protein divergence in birds are compared to previous estimates for other vertebrate taxa. At corresponding levels of the taxonomic hierarchy, birds consistently exhibit far smaller genetic distances than do many fishes and other vertebrates. [North American sparrows; Emberizidae; Fringillidae; protein evolution; electrophoresis; evolutionary rela-

Identification of Alfalfa Plants by Enzyme Electrophoresis1

The objective of this study was to apply the technique of starch gel electrophoresis to the identification of alfalfa (Medicago sativa L.) mother plants. These plants, preserved in field nursery plots for breeding and genetic studies, are subject to disease and pests. In many instances heavily diseased or dead plants are replaced by unidentified volunteers, thereby genetically contaminating the plots. Anodal isozyme peroxidases, esterases, and acid phosphatases from leaf tissue were very useful for “fingerprinting” each of the 21 mother plants tested: 18 tetraploids and three hexaploids. Zymograms were consistent for each plant when tested four times in a 3‐month span. The technique consisted of running crude extracts of apical meristems at 300 V for 3 hours on starch gels holding up to 16 different samples. After the run, the gels were sliced horizontally in three or four sections and each of these was assayed for a different enzyme. Starch gel electrophoresis is a simple, inexpensive, and reliable technique for the identification of alfalfa plants.

Genetic variation in deep-sea invertebrate populations: The bathyal gastropod Bathybembix bairdii

Five populations of the bathyal trochid gastropod Bathybembix bairdii (Dall) taken from depths of 579 to 1156 m in the Southern California Continental Borderland were surveyed for levels of genetic variability at 18 presumptive gene loci, using techniques of starch gel electrophoresis. All the populations were highly similar genetically; none of the populations possessed unique alleles. Four of 5 polymorphic loci scored in all the populations displayed statistically significant heterogeneity of allelic frequencies among stations. Only the diallelic fumarase (FUM) locus displayed a trend with depth. The geographic and depth patterns of the genetic variability in these populations do not support the hypothesis that the high levels of genetic variability observed in bathyal populations are an artifact of gene flow between populations differentiated with depth. The 16.2% of the loci heterozygous per individual of B. bairdii is of the same order as the average heterozygosities reported for other deep-sea invertebrate taxa. These reports of high genetic variability in a physically constant and seasonless environment are reviewed and discussed in the context of the trophic-stability hypothesis of Ayala and Valentine and the time, size and divergence hypothesis of Soule. Evidence is presented that despite the observed correlation of average heterozygosities in benthic marine invertebrates with seasonality of the trophic regime, this is not apparent for an enzyme system (leucine amino peptidase) which might be expected to display this trend if it reflected a genetic strategy to cope with trophic seasonality. The time, size and divergence hypothesis, focusing on population size, accounts for the general features of levels of genetic variability in deep-sea invertebrates.

Aberrant Gene Expression during the Development of Hybrid Sunfishes (Perciformes, Teleostei)

Summary Reciprocal intergeneric hybrid crosses were made between two species of centrarchids (Perciformes, Teleostei), the largemouth bass ( Micropterus salmoides ) (LMB) and the green sunfish ( Lepomis cyanellus ) (GSF). The LMB ♀ × GSF ♂ hybrid embryos underwent apparently normal morphological development. However, the hybrid embryos derived from the reciprocal GSF ♀ × LMB ♂ cross were morphologically abnormal, and all died near the time of hatching. Starch gel electrophoresis and specific histochemical staining techniques were utilized to investigate the ontogeny of enzymes encoded at each of 13 loci during the development of embryos of both parental species and both types of hybrids. Enzymes encoded in eight loci were present throughout the embryogenesis of one or both parental species. Enzymes encoded in these eight loci were also expressed throughout the development of both types of hybrid embryos. The electrophoretic mobilities of isozymes encoded in five of these eight loci were different for the two parental species. Although the maternal alleles at each of these five loci were expressed continuously during the development of both types of hybrid embryos, the parental alleles were not expressed until later in development. The five remaining loci were not expressed in the unfertilized eggs of either parental species, but were first expressed after fertilization. The onset of the expression of homologous loci was similar in embryos of the two parental species. During the development of the LMB ♀ × GSF ♂ hybrid embryos, the onset of gene expression for these five loci was delayed compared to that in the embryos of the two parental species. However, during the development of the GSF ♀ × LMB ♂ hybrid embryos, the onset of gene expression for most of these loci was earlier than that in the embryos of the two parental species. This precocious gene expression may reflect an aberrant pattern of gene regulation responsible for the abnormal development and lethality at hatching of the GSF ♀ × LMB ♂ hybrid embryos. A model is proposed to explain the various patterns of gene expression observed during embryogenesis of these reciprocal hybrids.

Variation and genetics of ribonucleases and phosphodiesterases in conifer seeds

Using techniques of starch gel electrophoresis, isoenzymes of ribonuclease 11 (RNase, EC 3.1.4.23) and phosphodiesterase I (PDase, EC 3.1.4.1) could be identified in endosperm tissue from dry seeds of three conifer species: Norway spruce (Picea abies). Scots pine (Pinus silvestris), and Douglas fir (Pseudotsuga menziesii). The RNase patterns mostly exhibited a relatively great number of isoenzyme bands as well as a considerable tree-to-tree variation, whereas the PDase system revealed only one enzyme zone in each of the three seed species. Furthermore, an isoenzyme variation within the PDase zones appeared to be very infrequent and could only be detected in Norway spruce and Douglas fir. However, the isoenzyme patterns of RNase as well as PDase showed significant differences between the conifer species. The genetic basis of the intraspecific isoenzyme variations could be easily analyzed, since the test material (seed endosperm) represented haploid tissues resulting from macrogametophytes after fertilization. Hence, it was possible to identify three polymorphic RNase gene loci in Douglas fir seeds, two in Scots pine seeds, and one in Norway spruce seeds. The PDase zone in each conifer species was controlled by one gene locus which revealed allelic forms only in Norway spruce and Douglas fir seeds.

Allelic expression and genetic distance in hybrid macaque monkeys

Levels of structural genic divergence at 21 loci encoding blood proteins were quantified in six macaque (Macaca) species, using standard techniques of starch-gel electrophoresis. Genetic distances between all pairs of species fall within a narrow range (0.080 less than or equal to D less than or equal to 0.250; D = 0.164) which is near the lower limit of genetic distances typically observed between other congeneric organisms. In an effort to measure levels of regulatory gene differences between these species, we have examined the patterns of allelic expression in their F1, F2, and backcross hybrids. Nine of the 21 loci examined encode allelic forms of the proteins with different electrophoretic mobilities in at least some of the individual parents of the hybrids. In all cases where expected, hybrids express fully both maternal and paternal allelic products, thus providing no strong evidence ofr a breakdown in the regulatory mechanisms responsible for proper expression of these genes. Results are compared to degrees of allelic repression previously observed in other hybrids, and are discussed within the context of current ideas about rates of regulatory gene evolution in mammals.

Improving of Starch Gel Electrophoresis Technique by Gamma Irradiation

AbstractElectrophoresis as well as starch gel technique are considered nowadays the best way of protein fractionation. This research was carried out towards improving that technique by modifying the gel properties by treating starch with gamma irradiation. Results indicated that gamma irradiation modified the starch molecules as its viscosity and its intrinsic viscosity were decreased and molecular breakdown and degradation occurred as well as modified amylose to amylopectin ratio and increased retrograded starch, leading to the improvement of the properties of the produced modified starch gel when compared to control sample.Protein electrophoresis confirmed this improvement because of the better fractionation of wheat gluten when the modified starch gel was used than with the control, as the modified starch gel was more transparent and flexible and had a longer distance of run than control sample. High molecular weight protein was able to migrate through modified starch gel contrary to unirradiated sample.

A preliminary genetic interpretation of the esterase isozymes of human tissues.

1. The esterase isozymes of human tissues have been investigated using the technique of starch-gel electrophoresis. Conventional naphthyl-azo dye linked stains and new fluorogenic staining methods were used to detect the isozymes. 2. Multiple isozymes were identified in every tissue and they were characterized in terms of their electrophoretic mobility, tissue distribution, developmental changes in utero, substrate specificity, inhibition properties and molecular weight. On these criteria 13 sets of esterase isozymes were identified, in addition to the esterase isozymes due to cholinesterase and carbonic anhydrase. 3. The data suggest that the 13 sets of isozymes are determined by at least nine different structural gene loci. 4. No electrophoretic variants were identified in a limited population survey of post-mortem tissues from adults and foetuses, except for the previously described esterase D (ESD) phenotypes.

DEEP-SEA ASTEROIDS: HIGH GENETIC VARIABILITY IN A STABLE ENVIRONMENT.

The distribution of geinetic variability in deep-sea organisms is virtually unknown, although estimates of genetic variability in the deep-sea fauna are directly relevant to some hypotheses concerning adaptive strategies in stable and unstable environments. One hypothesis, advanced to account for the great species diversity observed in the deep-sea, argues that deep-sea populations are highly specialized and possess little genetic variability (Grassle, 1972). Another hypothesis, also predicting that deep-sea populations should have little genetic variability, has been advanced by Bretsky and Lorenz (1969, 1970) to account for the waves of extinctions registered in the paleontological record. This hypothesis proposes that organisms living in stable environments should be genetically depauperate. Since the deep-sea provides one of the most stable environments in the world, both hypotheses can be tested by examining the amount of genetic variation in deep-sea organisms. In the course of a broad survey of genetic variability in diverse marine environments we have studied the genetic variability of four species of deep-sea asteroids from the San Diego Trough at a depth of 1,244 meters. Allelic variation at 24 individual loci coding for enzymes and other soluble proteins has been studied using the techniques of starch gel electrophoresis.

Polymorphism of the CA-I locus of carbonic anhydrase in baboon.

Polymorphism of erythrocytic carbonic anhydrase was studied by means of the usual technique of starch gel electrophoresis in Papio papio, Papio hamadryas, Papio cynocephalus and Papio anubis. In a sample containing both Papio cynocephalus and Papio anubis, examples of the homozygote CA-Ib/CA-Ib were found. A study of parental transmission established the CA-Ib allelic form.

Genetic Distance Among Certain Species ofMucor

A comparison of homologous enzymes in mycelial extracts of 20 species of Mucor was made using starch gel electrophoresis and staining techniques for 11 different enzymes. On the basis of genetic distance, a computerized cluster analysis was used to arrange the species into groups reflecting quantitative phylogenetic relationships among the examined species Conclusions are drawn about the validity of present concepts of Mucor phylogenetics.

Electrophoretic types of transaldolase, transketolase, and other enzymes in bifidobacteria.

The technique of starch-gel electrophoresis with staining for transaldolase, transketolase, 6-phosphogluconate dehydrogenase, and aldolase, was used to compare 49 representative strains of the genus Bifidobacterium, the deoxyribonucleic acid homology relationships of which were known. The zymograms obtained with fructose-6-phosphate as substrate for staining were also recorded and compared. Parallel experiments were made with spectrophotometric techniques to evaluate the specificity of the staining for transaldolase and transketolase. In each of the enzymatic activities investigated, isozymes were revealed by this technique. Their distribution is discussed.

An analysis of five enzyme‐gene loci in four etheostomid species (Percidae: Pices) in an area of possible introgression

The possibility of natural hybridization between two species of darters, Etheostoma spectabile and caeruleum was investigated using the technique of starch gel electrophoresis of skeletal muscle homogenates. Inter‐ and intraspecific genetic variability at five enzyme loci was found. Presumed morphological hybrids are most probably not genetic hybrids as judged by their enzyme genotypes. The analysis of enzyme variability supports a previously published phylogeny for the darters.

Enzyme variability in the Drosophila Willistoni group. V. Genic variation in natural populations of Drosophila equinoxialis.

SUMMARYWe have studied genetic variation at 27 loci in 42 samples from natural populations of a neotropical species,Drosophila equinoxialis, using standard techniques of starch-gel electrophoresis to detect allelic variation in genes coding for enzymes. There is considerarle genetic variability inD. equinoxialis. We have found allelic variation in each of the 27 loci, although not in every population. On the average, 71% of the loci are polymorphic – that is, the most common allele has a frequency no greater than 0·95 – in a given population. An individual is heterozygous on the average at 21·8% of its loci.The amount of genetic variation fluctuates widely from locus to locus. At theMdh-2locus arout 1% of the individuals are heterozygotes; at the other extreme more than 56% of the individuals are heterozygous at theEst-3. At any given locus the configuration of allelic frequencies is strikingly similar from locality to locality. At each and every locus the same allele is generally the most common throughout the distribution of the species. Yet differences in gene frequencies occur between localities. The pattern of genetic variation is incompatible with the hypothesis that the variation is adaptively neutral. Genetic variation inD. equinoxialisis maintained by balancing natural selection.The amount and pattern of genetic variation is similar inD. equinoxialisand its sibling species,D. willistoni. Yet the two species are genetically very different. Different sets of alleles occur at nearly 40% of the loci.

A Mutant A-Type Lactate Dehydrogenase Subunit in Fundulus heteroclitus (Pisces: Cyprinodontidae)

The rarity of mutant alleles at the loci coding for the polypeptide subunits of the tetrameric enzyme lactate dehydrogenase (LDH) is emphasized in all vertebrate classes. Fundulus heteroclitus synthesizes three physicochemically distinct types of LDH subunits (A, B and E) under the direction of three genetic loci. Two codominant alleles are present at the B locus which code for the synthesis of two B-type subunits (B and B'). Antisera specific to fish LDH subunits selectively precipitate LDH isozymes from tissue homogenates of F. heteroclitus. Coupled with starch gel electrophoresis and enzyme specific staining techniques, it is possible to determine the LDH isozyme composition of tissues and the genetic interrelationships of the isozymes. Employing this procedure, we have discovered a rare A-locus allele coding for a subunit designated A'. At alkaline pH's, the A' subunit is less negatively charged than the A subunit and migrates more slowly toward the anode. This allele was found only in one out of more than 300 specimens examined. This specimen was an AA' heterozygote.

Comparative zone electrophoresis of enzymes of Pseudomonas solanacearum and Pseudomonas cepacia.

The technique of starch-gel electrophoresis with specific staining for a series of enzymes was used to compare 21 Pseudomonas strains representing both P. cepacia and P. solanacearum. These experiments produced no evidence for close similarity of the two species. Twelve strains of P. solanacearum were compared by means of data obtained from nine different enzymes, and the data indicate that these strains belong in two biotypes. Except for the assignment of two strains, these groups are the same as the two major groups previously derived from nutritional properties and from deoxyribonucleic acid hybridization experiments. Eleven enzymes were available for comparisons of the P. cepacia strains. Eight of these strains form a homogeneous group, but the last strain, number 249, differs considerably from the other representatives of the species.

Turnover of genetically defined catalase isozymes in maize.

The turnover of two homotetramers of catalase, V(4) and Z(4), specified by genes at two separate loci, has been studied during germination of maize seed by the techniques of density labeling and starch-gel electrophoresis. Both isozymes were shown to be turning over during this time. However, Z(4) accumulates because the rate of synthesis exceeds the rate of degradation, whereas V(4) slowly disappears because the rate of degradation exceeds the rate of synthesis. Evidence is also presented that the rate of synthesis of Z(4) exceeds that of V(4), which suggests that this may be a major factor in the differential expression of the two catalase genes.

Adenylate kinase isoenzyme patterns in tissues of people of different phenotype.

Three distinct isoenzyme sets have been demonstrated for adenylate kinase (AK) from human tissues by the technique of starch gel electrophoresis. In adult and fetal muscle, liver, kidney, brain, spleen, lung, and leukocytes the same set was found, though individual bands had different intensities. In adult red cells and in fetal red cells separate and distinctive isoenzyme sets were observed. However, in all tissues, adult and fetal, the phenotype of an individual was recognizable by the presence or absence of a characteristic cathodal band. The apparent anomaly of the same phenotype being expressed in different isoenzyme sets was resolved by the demonstration that in adult and fetal red cells AK complexed with hemoglobin. The implications of this complex are briefly discussed.