Tissue-specific and dietary control of alpha-amylase gene expression in the adult midgut of Drosophila melanogaster.

The regulatory effects of allelic substitution at the trans-acting mapP locus and of dietary glucose on the expression of the duplicate genes for alpha-amylase (Amy) in Drosophila melanogaster were examined in the anterior midgut and posterior midgut regions of mature flies. The levels of amylase activity and amylase protein, as well as the abundance of amylase-specific RNA, were quantified. All 3 parameters of Amy expression were concordant. Results indicate that the effects of both mapP and dietary glucose are exerted at the level of amylase RNA. However, the tissue-specific effects of mapP are restricted to the posterior midgut and can therefore be distinguished from the effects of glucose in food medium, which influences amylase RNA levels in both the anterior and posterior midgut regions. Our data suggest that, in large part, strain-specific effects of dietary glucose can be explained on the basis of alternate alleles at the mapP locus in different homozygous strains of flies. Levels of amylase RNA in tissue extracts of flies from an amylase-null strain were also measured. Low levels were observed in both anterior and posterior midgut extracts. These were unresponsive to dietary conditions.     

Population Genetics of Drosophila Amylase. I. Genetic Control of Tissue-Specific Expression in D. PSEUDOOBSCURA

Drosophila pseudoobscura is polymorphic for tissue-specific expression of alpha-amylase in adult midguts. This enzyme is encoded by a single locus, Amy, on the third chromosome. In this paper we show: (1) Up to about 12 days post-eclosion, the midgut activity patterns remain stable; after 12 days areas not showing activity previously begin to show activity. Thus, the genes controlling the expression of Amy are temporally acting. (2) Diet affects the quantitative, but not the qualitative, expression of Amy. (3) The expression of Amy in adult midguts is under genetic control. Selection for different frequencies of patterns is possible; realized heritabilities are 0.20 to 0.50. Partial linkage with third chromosome inversions has been demonstrated; the genes or elements controlling Amy expression are not, however, confined to the third chromosome. (4) The genetic elements controlling tissue-specific expression of amylase do not coordinately control the expression of five other "digestive-type" enzymes that were studied.--This polymorphism appears to be analogous to that studied by Abraham and Doane (1978) in D. melanogaster, wherein they have mapped regulatory genes.     

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.     

Dosage compensation and dietary glucose repression of larval amylase activity inDrosophila miranda

The functional locus for alpha-amylase (Amy) in Drosophila miranda is in the evolutionarily new X2 chromosome. X2 evolved from an autosome in response to an ancestral autosome-Y translocation that gave rise to the "neo-Y" chromosome of this species. Y-linked Amy, if still present in the ancestrally translocated element, is unexpressed. Dosage compensation for amylase activity was examined in larvae of the S 204 strain. Since dietary glucose is known to repress Amy expression in Drosophila melanogaster, dosage compensation of amylase activity in male larvae of D. miranda was tested by rearing larvae of both sexes on yeast diets with or without a glucose supplement. The WT 10 strain of Drosophila persimilis, a sibling species in which Amy is autosomally linked, was used as a reference for tests of amylase activity differences between the sexes. On the diet with glucose, Amy expression was repressed in both WT 10 and S 204 larvae and male larvae of S 204 displayed dosage compensation for amylase activity. On the nonrepressing diet consisting of yeast alone, S 204 continued to display dosage compensation.     

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.     

Signaling by intracellular Ca2+ and H+ in larval mosquito (Aedes aegypti) midgut epithelium in response to serosal serotonin and lumen pH

The midgut of larval mosquitoes (Aedes aegypti) mediates a cycle of alkali secretion in the anterior segment (AMG) followed by partial reacidification in the posterior segment (PMG); both processes are serotonin-dependent. Here we report that intracellular Ca(2+)(Ca(i)(2+)) as indicated by Fura-2 fluorescence, is elevated in both tissues in response to serotonin, but the time courses differ characteristically in the two gut segments, and Ca(2+)-free solution abolishes the serotonin response in AMG, but not in PMG, whereas Thapsigargin, an inhibitor of endoplasmic Ca(2+) transport, abolished responsiveness to 5-HT in PMG. These results suggest the origins for the Ca(2+) signal differ between the two tissues. Quantitative real-time RT-PCR revealed expression of 5 putative 5-HT receptor types in AMG, including 5-HT(2)-like receptors which would be expected to initiate a Ca(2+) signal. None of these receptors were highly expressed in PMG. Cyclic AMP (cAMP) is a secretagogue for both tissues, but H89, an inhibitor of Protein Kinase A (PKA), is also a secretagogue, suggesting that the stimulatory effect of cAMP involves a non-PKA pathway. Cytochalasins B and D block the effect of 5-HT in AMG, suggesting a vesicle-fusion mechanism of activation of the basal V-ATPase in this tissue. Finally, in PMG, elevation of luminal pH increases (Ca(i)(2+)) and decreases intracellular pH as measured by BCECF fluorescence. These responses suggest that the rate of acid secretion by PMG might be responsive to local demand for luminal reacidification as well as to serosal serotonin.     

RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus

In the Mediterranean field cricket, Gryllus bimaculatus, the action of sulfakinin (SK) gene expression on food intake, food transport in the gut and carbohydrate digestion (alpha-amylase activity) was investigated by using the RNA interference (RNAi) method. Injection of SK double-stranded (ds) RNA into the abdomen of female adults and last instar larvae led to a systemic silencing of the SK gene, as was shown by RT-PCR studies. In adults, suppression of SK gene expression was effective from the first day after injection up to at least the third day. Treatment of the adult crickets by injection or feeding of dsRNA led to a stimulation of the food intake. Assuming that the gene silencing is followed by a depletion of the SK in tissues and/or haemolymph implies an inhibitiory role of the native SK peptides on food intake. The alpha-amylase activity in vitro in the midgut tissue and in the secretions of adult females was not affected by silencing the SK gene.     

Morphological aspects of blood digestion in a parasitic mite Bakericheyla chanayi

All life stages of B. chanayi (Acariformes: Cheyletidae) are characterized by occasional bloodsucking and a long period of digestion. No newly engorged mites were found during the period of their host birds' migration. The fine structure of the digestive tract of a blood-feeding acariform mite is described for the first time. The anterior midgut (AMG) is a place of blood digestion, while the posterior midgut (PMG) is involved in nitrogen metabolism forming guanine crystals as the main end-product. The AMG epithelium consists of digestive cells that probably arise from mitotically active basal cells with high synthesizing activity.As observed in ticks, blood digestion is accompanied by the formation of huge endosomes that serve as places of storage and sorting of ingested material. Digestive cells show different types of endocytotic activity as well as various late endosomes, which implies different subcellular pathways for different blood components. In both midgut regions, elimination of the excretory material occurs by apocrine secretion or by discharging of apical cell fragments (loaded with lysosomes) into the gut lumen. The formation of guanine granules occurs inside the lysosomes of PMG epithelial cells thus having much in common with intracellular digestion. Peculiarities of intracellular blood digestion were analyzed according to the modern hypothesis of endocytosis and compared to what is known in ticks.     

Investigation of the midgut structure and ultrastructure in <Emphasis Type="Italic">Cimex lectularius</Emphasis> and <Emphasis Type="Italic">Cimex pipistrelli</Emphasis> (Hemiptera: Cimicidae)

Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions—the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.     

Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura

The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far.      

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.     

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.     

Barley alpha-amylase genes. Quantitative comparison of steady-state mRNA levels from individual members of the two different families expressed in aleurone cells

We have cloned and sequenced two barley alpha-amylase genes belonging to the high pI isozyme family, one of which, Amy6-4, corresponds to a cDNA previously characterized by our laboratory. A 750-base pair probe from Amy6-4, representing primarily the promoter/upstream sequences cross-hybridizes on genomic Southern blots under stringent conditions to five other genes or pseudogenes; this demonstrates that the promoter/upstream region in these different members of the gene family is highly conserved. In contrast, this probe hybridizes very poorly to the genomic fragment containing the other cloned high pI gene, Amy46, a finding consistent with substantial divergence of sequence about 200 base pairs upstream from the TATA box of each. We compared steady-state mRNA levels from these individual genes to levels for mRNAs from two low pI alpha-amylase genes and from the single copy gene for aleurain, a thiol protease, using quantitative S1 nuclease protection assays. We found, in RNA from aleurone cells treated with gibberellic acid for 19-24 h, that the two low pI alpha-amylase mRNAs are each about five times more abundant than Amy6-4 or aleurain, which are, in turn, about 10 times more abundant than Amy46. These results indicate that as many as seven closely related high pI genes are needed to provide mRNA levels approaching those from the two low pI genes. We speculate that the substantially lower level of expression of Amy46 may be related to its divergent sequence upstream from the promoter.     

DNA rearrangement causes multiple changes in gene expression at the amylase locus inDrosophila melanogaster

A spontaneous null mutation at the alpha-amylase locus in Drosophila melanogaster was recovered from a laboratory population. The mutant strain was found to lack amylase enzyme production and to produce low, but detectable, levels of amylase mRNA. Moreover, the null strain is also lacking the glucose repression of amylase mRNA production which is seen in wild-type strains. The mutant phenotype correlates with a rearrangement in genomic DNA which, in turn, corresponds to a simple inversion in the arrangement observed most frequently in North American populations of D. melanogaster, including the common laboratory strain, Oregon-R. These results have implications for our understanding of both the evolution of the duplicated amylase gene structure and the regulation of amylase gene expression.