Restriction map and alpha-amylase activity variation among Drosophila mutation accumulation lines

The specific activities of alpha-amylase were measured for two sets of mutation accumulation lines, each set having originated from a different lethal-carrying second chromosome and SM1(Cy) chromosome and having been maintained by a balanced lethal system for about 300 generations. Significant variation was found to have accumulated among lines of both sets. Because of dysgenic crosses in the early generations of mutation accumulation, insertions or deletions of transposable elements in the Amy gene region were suspected of being the cause of this variation. In order to test this possibility, the structural changes in the 14 kb region of these chromosomes that includes the structural genes for alpha-amylase were investigated by restriction map analysis. We found that most part of the activity variation is due to replacements of a chromosomal region of SM1(Cy), including the structural genes for alpha-amylase, by the corresponding regions of the lethal chromosomes. One line also contained an insertion in this region but this line has an intermediate activity value. Thus, insertions of transposable elements into the Amy gene region were not found to be responsible for the new variation observed in alpha-amylase activity. If we remove those lines with structural changes from the analysis, the genetic variance of alpha-amylase specific activity among lines becomes non-significant in both sets of chromosomes.     

Genetic Localization and Action of Regulatory Genes and Elements for Tissue-Specific Expression of alpha-Amylase in DROSOPHILA MELANOGASTER

Regulation of tissue-specific alpha-amylase (Amy) expression in Drosophila melanogaster was investigated with a newly developed method that detects the distribution of alpha-amylase allozymes in midguts of single adults or third-instar larvae. Trans regulation was found for alpha-amylase production in the posterior midgut (PMG) of adults, whereas cis regulation was demonstrated for the larval midgut. Independent regulation of components of the duplicated Amy locus was found in larvae. Recombination between the components of the Amy locus did not result in separation of the putative, very closely linked (less than 0.1 cM) cis-acting regulatory elements for alpha-amylase expression in the anterior midgut (AMG) of larvae. The expression of one of the components of the duplicated Amy locus in the AMG of larvae was influenced by a regulatory gene that was mapped at 2-79.1. alpha-Amylase expression in the adult PMG was controlled by a trans-acting regulatory gene localized at 2-79.0, in agreement with the data of Abraham and Doane.     

Structural organization of the Amy locus in seven strains of Drosophila melanogaster.

Restriction maps were made by Southern blot analysis of the Amy (alpha-amylase) region in 7 strains of D. melanogaster using endonucleases SalI, XhoI and EcoRI. These were compared to the map of lambda Dm65 which contains the cloned Amy region. Strains used produce either two amylase variants, a single variant, or no amylase, yet all 7 strains carry two Amy genes as inverted repeats at the Amy locus. This and the orientation of the repeats resembles the situation in lambda Dm65. Most restriction sites mapped are conserved but two strains contain a large insertion which differs in size and position between strains. A complex anomaly, probably an inversion, exists at the Amy locus in a null strain. Maps for our Amy1,3 strain and the lambda Dm65 clone are identical, the DNA of each having been derived from a Canton-S wild stock. Restriction and genetic maps of the Amy region were aligned and alleles assigned to the proximal and distal genes, Amy-p and Amy-d.     

The Genetic Structure of Natural Populations of Drosophila Melanogaster. Xxii. Comparative Study of DNA Polymorphisms in Northern and Southern Natural Populations

Restriction map variation in four gene regions (Adh, Amy, Pu and Gpdh) was surveyed for 86 second chromosomes from northern (Aomori) and southern (Ogasawara) Japanese populations of Drosophila melanogaster (43 chromosomes from each population). The regions examined cover a total of 62 kilobases. Estimates of nucleotide diversity (pi) were approximately constant across the gene regions and populations examined. The distribution of restriction site polymorphisms was compatible with the expectation from the neutral mutation-random genetic drift hypothesis, but insertion/deletion polymorphisms were not consistent with it. While the two populations shared a majority of restriction site polymorphisms, frequencies of individual restriction site variants were significantly different between the two populations at 7 out of 35 segregating sites. In addition, an insertion in the Amy region was found in 15 chromosomes from the Ogasawara sample but absent in the Aomori sample. A considerable difference was observed in the number of rare insertions and deletions between the two populations. The numbers of aberrations uniquely represented were 16 in the Ogasawara sample and only 3 in the Aomori sample. These findings suggest that the two populations were differentiated from each other to some degree by means of random genetic drift and/or other factors.     

Developmental Variation in Amylase Allozyme Activity Associated with Chromosome Inversions in DROSOPHILA PERSIMILIS

The amylase locus in Drosophila persimilis is polymorphic for allozymes, two of which show associations with naturally occurring chromosome 3 inversions. Amy1.09 occurs at high frequencies only in Whitney (WT), while the other common arrangements-Standard (ST), Klamath (KL) and Mendocino (MD)-are predominantly Amy 1.00. We have examined numerous strains, representing various electromorphs and inversions, for variation in cis-specific activity expression in both third-instar larvae and adults. Comparisons of these two life stages also allows the survey of developmental variation in amylase activities. The amount of activity variation exceeds electrophoretic variation at this locus. Moreover, this variation is largely nonrandom and reveals more genic divergence among inversions. The 1.00 allozyme of MD is more active than 1.00 KL in larvae and adults and shows a different developmental pattern. The activity of the 1.00 allozyme of KL is greater than 1.00 allozyme of ST in larvae and adults, but these two arrangements have similar developmental patterns. WT 1 with a 1.00 allele is dramatically different from the 1.00 allozymes of other arrangements in its developmental pattern. The 1.09 allozymes has high activity in WT and KL, but these arrangements differ in their developmental pattern of expression, WT being more active in adults. F2 segregational analyses are consistent with the variation being due to either structural enzyme variants or closely linked cis-acting regulatory elements. We argue that the suppression of recombination between arrangements has allowed the divergence in amylase activity among inversions.     

Genetic coadaptation of the amylase gene system in Drosophila melanogaster: evidence for the selective advantage of the lowest AMY activity and of its epistatic genetic background

In natural populations of Drosophila melanogaster, an amylase isozyme with the lowest alpha-amylase activity (AMY(1,1)) is predominant. To evaluate the selective significance of AMY(1,1) and its regulatory factor(s), we examined selection experiments in laboratory populations on two distinct food environments. After 300 generations, AMY(1,1) became predominant (89%) in a glucose (a product of AMY)-rich environment, while an isozyme with higher alpha-amylase activity, AMY(1,6), became predominant (83%) in a starch (substrate)-rich environment. We found that the identical alleles of the amylase (Amy) gene, which encodes each of AMY(1,1) and AMY(1,6), were shared between the two populations in the different food environments, employing the nucleotide sequencing of the duplicated Amy genes. Nevertheless, AMY(1,6) homozygotes selected in the starch-rich environment had a twofold higher AMY enzyme activity than those selected in the glucose-rich environment, suggesting a coadaptation of the coding region and its regulatory factor(s) on the genetic background. Such a difference in AMY enzyme activity was not detected between AMY(1,1) homozygotes, suggesting that the effect of the genetic background is epistatic. Our results indicate that natural selection is working on the Amy gene system as a whole for flies to adapt to the various food environments of local populations.     

Cloning and expression of the alpha-amylase gene from Bacillus stearothermophilus in several staphylococcal species

The plasmid-coded alpha-amylase gene of Bacillus stearothermophilus (amy) was cloned in Staphylococcus carnosus using plasmid pCA43 as a vector. The amy gene was located on a 5.4-kb HindIII DNA fragment of the hybrid plasmid pamy7. When transformed into other staphylococcal species, plasmid pamy7 exhibited marked differences in the production of alpha-amylase (alpha Amy). Most active for heterospecific alpha Amy production was Staphylococcus aureus. In its culture supernatant nearly half as much alpha Amy activity was found as for the donor strain B. stearothermophilus. All staphylococcal species were able to secrete alpha Amy, since more than 80% of the enzyme activity was found in the culture supernatant. The extracellular alpha Amy of S. aureus [pamy7] was purified to homogeneity. The enzyme exhibited an Mr of approx. 58 000, an optimum activity at pH 5.3-6.3 and at 65 degrees C. Although the enzyme was stable at 65 degrees C for at least 3 h, its thermostability was not unusual. The enzymatic properties of the alpha Amy from S. aureus were similar to those previously reported for various B. stearothermophilus strains.     

Use of the thermoinducible temperate phage Phi 105cts139 for cloning in Bacillus subtilis

The gene for alpha-amylase from Bacillus amyloliquefaciens having a foreign promoter providing gene expression in logarithmic growth phase and the cat gene of plasmid pC194 (AC fragment) were inserted into thermoinducible prophage phi 105 cts139. Possibility of amylolytic activity enhancement was studied after thermoinduction. When AC fragment and random PstI restricts of phage DNA were ligated and used to transform Bacillus subtilis 1A289 (phi 105 cts139) the Amy+ CmR transformants were obtained having the different levels of increased amylolytic activity (maximum--26 fold). Numerous phages without insert found in induced lysates suggest that insertions were unstable and (or) the clones were double lysogens for hybrid and original type phages. Stable insertion of AC fragment replacing the PstI-H-fragment of phage DNA revealed that all Amy+ CmR transformants were double lysogens. Inducibility depended on the insertion orientation.     

Population Genetics of Drosophila Amylase. II. Geographic Patterns in D. PSEUDOOBSCURA

Morph frequencies of three related polymorphisms were determined in ten natural populations of Drosophila pseudoobscura. They are the well-known inversion polymorphism of the third chromosome and the polymorphism for alpha-amylase produced by the structural gene Amy (which resides on the third chromosome). The third polymorphism was for tissue-specific expression of Amy in adult midguts; a total of 13 different patterns of activity have been observed. The preceding paper (Powell and Lichtenfels 1979) reports evidence that the variation in Amy expression is under polygenic control. Here we show that the polymorphism for midgut patterns occurs in natural populations and is not an artifact of laboratory rearing.--From population to population, Amy allele frequencies and frequencies of inversions belonging to different phylads vary coordinately. The geographic variation in alpha-amylase midgut activity patterns is uncorrelated with that for the other two types of polymorphisms. Furthermore, no correlation was detected between activity pattern(s) and Amy genotype(s) when both were assayed in the same individual.--These results imply that whatever the evolutionary-ecological forces are that control frequencies of the structural gene variants, they are not the same factors that control the frequencies of polymorphic genetic factors responsible for the tissue-specific expression of the enzyme.     

Molecular and Phenotypic Variation of the Zw Locus Region in Drosophila Melanogaster

Restriction map polymorphism in a 13-kb region of the Zw locus in Drosophila melanogaster was investigated for 64 X chromosome lines with seven 6-cutter and ten 4-cutter restriction enzymes. A total of 203 restriction sites were scored, of which 20 were found to be polymorphic. The estimated nucleotide variation for this region for overall data (pi = 0.003 and 0.001, and theta = 0.003 and 0.002, for 4-cutter and 6-cutter studies, respectively) was smaller than that reported for most regions studied in D. melanogaster. It was found that the Slow allozyme has a larger nucleotide variation and haplotype diversity than the Fast allozyme. Results suggest the relatively recent divergence of the Fast allozyme from the Slow allozyme. Glucose 6-phosphate dehydrogenase (G6PD) activity was measured as a phenotype of the Zw locus. A significant difference in G6PD activity between allozymes was detected. The between-line effect was highly significant within the Slow allozyme, but was not significant within the Fast allozyme. Although a direct causative link could not be established, these results suggest an association between the amounts of quantitative and molecular genetic variation at the Zw locus region.     

Substrate-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments

The ragi alpha-amylase/trypsin bifunctional inhibitor (RATI) from Indian finger millet, Ragi (Eleucine coracana Gaertneri), represents a new class of cereal inhibitor family. It exhibits a completely new motif of trypsin inhibitory site and is not found in any known trypsin inhibitor structures. The alpha-amylase inhibitory site resides at the N-terminal region. These two sites are independent of each other and the inhibitor forms a ternary (1:1:1) complex with trypsin and alpha-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the alpha-amylase inhibition kinetics is a complex one if larger (> or =7 glucose units) substrate is used. While a complete inhibition of trypsin activity can be achieved, the inhibition of amylase is not complete even at very high molar concentration. We have isolated the N-terminal fragment (10 amino acids long) by CNBr hydrolysis of RATI. This fragment shows a simple competitive inhibition of alpha-amylase activity. We have also synthesized various peptides homologous to the N-terminal sequence of RATI. These peptides also show a normal competitive inhibition of alpha-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of alpha-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to alpha-amylase and its implications on the mechanism and kinetics of inhibition.     

A Comprehensive Study of Genic Variation in Natural Populations of Drosophila Melanogaster. IV. Mitochondrial DNA Variation and the Role of History Vs. Selection in the Genetic Structure of Geographic Populations

Preliminary studies with restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) in natural populations of Drosophila melanogaster revealed considerable variation in terms of nucleotide sequence and overall size. In this report we present data from more isofemale lines and more restriction enzymes, and explore the utility of the data in inferring a colonization history of this species. Size variation in the noncoding A + T-rich region is particularly plentiful, with size variants occurring in all restriction site haplotypes in all populations. We report here classes of small-scale mobility polymorphisms (apparent range of 20 bp) in specific restriction fragments in the coding region. The variation in one such fragment appears to be generated even more rapidly than in the noncoding region. On the basis of the distribution of restriction site haplotypes, the species range can be divided into three major regions along longitudinal lines: Euro-African populations are the most diverse and are taken to be oldest; Far East populations have a complex distribution of haplotypes; Western Hemisphere populations are the least diverse and are interpreted to be the youngest. The history inferred from mtDNA alone is remarkably similar to one based on several nuclear markers. The mtDNA haplotype distribution is also very different from that of allozymes in these same populations. We interpret this as further evidence that natural selection is still the most parsimonious explanation for the parallel latitudinal allozyme clines in this species.     

Insertion-deletion variation at the yellow-achaete-scute region in two natural populations of Drosophila melanogaster

We have surveyed the region of the X chromosome of Drosophila melanogaster which encodes the yellow, achaete and scute genes for restriction map variation. Two natural populations, one from North Carolina, U.S.A. and the other from southern Spain were screened for variation at about 70 restriction sites and for variation due to DNA insertion or deletion events in 120 kilobases of DNA. Mean heterozygosity per nucleotide was estimated to be 0.0024 and 15 large insertions were found in the 49 chromosomes screened. Extensive disequilibrium between polymorphic sites were found across much of the region in the North Carolina population. The frequency of large insertions, which usually correspond to transposable genetic elements, is significantly lower than has been observed in autosomal regions of the genome. This is predicted for X-linked loci by certain models of transposable element evolution, where copy number is restricted by virtue of the recessive deleterious effects of the insertions. Our results appear to support such models. The deficiency of insertions may in this case be enhanced by hitch-hiking effects arising from the high level of disequilibrium.