An investigation on protein and amino acid contents in scales and muscles of pomfret Parastromateus niger (Bloch, 1795) and Pampus argenteus (Eupharasen, 1788).

The present investigation was aimed to examine the percentage quantity of protein and amino acids in scales and muscles of Pampus argenteus and Parastromateus niger gathered from the local fish market of district Quetta of Balochistan. About 80 specimens of these two species, i.e., Pampus argenteus (N=40) and Parastromateus niger (N = 40), were collected from April 2017 to May 2018. In general, crude protein content was high in scales, that is, 71.03% in Parastromateus niger and 52.11% in Pampus argenteus, as well as in muscles of two Pomfret species of fishes i.e., 63.44% in Pampus argenteus and 60.99% in Parastromateus niger on a dry-weight basis, respectively. Likewise, the muscles and scales of Parastromateus niger reveal well compositions of amino acids that include proline was found to be high, and methionine was less than other amino acids, whereas threonine was found high in the scales of Pampus argenteus, but methionine was observed in lesser amount. However, the amino acids found in Pampus argenteus muscles also showed different compositions, such as lysine was found to be high, but histidine was less, respectively. In comparison, amino acids like tryptophan and cysteine were not detected in both scales and muscles of these Pomfret species of fishes. Thus, this study was based on analyzing the utilization of both Pomfret species of scales and meat whether they could have values as good supplements of both protein and certain kinds of essential amino acids in animal diets.

Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan.

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-A resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily beta-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

Divergent decapentaplegic expression patterns in compound eye development and the evolution of insect metamorphosis.

In the fruit fly Drosophila, the patterning genes decapentaplegic and wingless contribute to the spatial control of retina development in an antagonistic manner. We examined the expression patterns of these genes in the developing visual system of the hemimetabolous grasshopper Schistocerca americana and the primitive holometabolous beetle species Tribolium castaneum. The pattern of wingless expression was strongly conserved as a pair of lateral domains at the anterior margins of both the developing retina and the developing optic lobes. The expression of decapentaplegic, on the other hand, is different. Unlike in Drosophila, no decapentaplegic expression was detected before the onset of photoreceptor differentiation in the retinal precursor tissue of either grasshopper or beetle. Moreover, the subsequent expression of decapentaplegic in the latter species was not concentrated in the moving front of retina differentiation, as in Drosophila, but observed in anterior and posterior regions. Our results indicate that Drosophila eye development contains elements of both ancestral and derived regulatory gene functions. The requirement for decapentaplegic as an antagonist of wingless during the early development of the Drosophila retina might have originated during the evolution of insect metamorphosis.

Genetic suppression of polyglutamine toxicity in Drosophila.

A Drosophila model for Huntington's and other polyglutamine diseases was used to screen for genetic factors modifying the degeneration caused by expression of polyglutamine in the eye. Among 7000 P-element insertions, several suppressor strains were isolated, two of which led to the discovery of the suppressor genes described here. The predicted product of one, dHDJ1, is homologous to human heat shock protein 40/HDJ1. That of the second, dTPR2, is homologous to the human tetratricopeptide repeat protein 2. Each of these molecules contains a chaperone-related J domain. Their suppression of polyglutamine toxicity was verified in transgenic flies.

Preventing neurodegeneration in the Drosophila mutant bubblegum.

The Drosophila melanogaster recessive mutant bubblegum (bgm) exhibits adult neurodegeneration, with marked dilation of photoreceptor axons. The bubblegum mutant shows elevated levels of very long chain fatty acids (VLCFAs), as seen in the human disease adrenoleukodystrophy (ALD). In ALD, the excess can be lowered by dietary treatment with "Lorenzo's oil," a mixture of unsaturated fatty acids. Feeding the fly mutant one of the components, glyceryl trioleate oil, blocked the accumulation of excess VLCFAs as well as development of the pathology. Mutant flies thus provide a potential model system for studying mechanisms of neurodegenerative disease and screening drugs for treatment.

Extended life-span and stress resistance in the Drosophila mutant methuselah.

Toward a genetic dissection of the processes involved in aging, a screen for gene mutations that extend life-span in Drosophila melanogaster was performed. The mutant line methuselah (mth) displayed approximately 35 percent increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat, a free-radical generator. The mth gene predicted a protein with homology to several guanosine triphosphate-binding protein-coupled seven-transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span.

Dual functions of the Drosophila eyes absent gene in the eye and embryo.

In eyes absent (eya) mutants, eye progenitor cells undergo cell death early in development. Whereas the phenotype of eya1 is limited to the eye, other mutations are lethal. Genetic and molecular analysis reveals that mutations in one region of the gene cause embryonic lethality, whereas mutations throughout the gene cause defects in eye development. Mosaic analysis indicates that the eya requirement is cell autonomous. In eye-specific mutants, expression in the eye disc is lacking while embryonic expression is normal. Both the type I and type II transcripts are expressed in the developing eye, and expression of either can rescue the eye phenotype. These data indicate a specific requirement for eya function in eye progenitor cells that is normally fulfilled by both transcripts.

Multiple roles of the eyes absent gene in Drosophila.

The eyes absent (eya) gene plays an essential role in the events that lead to formation of the Drosophila eye; without expression of eya in retinal progenitor cells, they undergo programmed cell death just prior to the morphogenetic furrow, leading to an eyeless or reduced eye phenotype. The eya gene has recently been found to be highly conserved to humans, defining a new gene family. Insights into the gene's function in the fly, therefore, are likely to be relevant to the role of its homologs in vertebrates. Detailed studies at the subcellular level indicate that the Eya protein is localized to the nucleoplasm, suggesting a role in control of nuclear events. The eya gene shows expression and roles in tissues other than the eye, including subsets of cells of the adult visual system, brain, and ovary, as well as an elaborate expression pattern in the embryo. Various mutations in the eya gene cause loss of ocelli, female sterility, or lethality. Analysis of the embryonic lethal phenotype indicates that mutant alleles show defects in head morphogenesis. These data indicate that eya has critical roles in morphogenesis of a number of tissues in the animal, in addition to its role in early eye formation. Despite multiple roles at multiple stages of development of the fly, both the type I and type II forms of the protein, when expressed ectopically during larval development, can direct eye formation.

Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death.

Wolbachia, a maternally transmitted microorganism of the Rickettsial family, is known to cause cytoplasmic incompatibility, parthenogenesis, or feminization in various insect species. The bacterium-host relationship is usually symbiotic: incompatibility between infected males and uninfected females can enhance reproductive isolation and evolution, whereas the other mechanisms enhance progeny production. We have discovered a variant Wolbachia carried by Drosophila melanogaster in which this cozy relationship is abrogated. Although quiescent during the fly's development, it begins massive proliferation in the adult, causing widespread degeneration of tissues, including brain, retina, and muscle, culminating in early death. Tetracycline treatment of carrier flies eliminates both the bacteria and the degeneration, restoring normal life-span. The 16s rDNA sequence is over 98% identical to Wolbachia known from other insects. Examination of laboratory strains of D. melanogaster commonly used in genetic experiments reveals that a large proportion actually carry Wolbachia in a nonvirulent form, which might affect their longevity and behavior.

Glia in the chiasms and medulla of the Drosophila melanogaster optic lobes.

Different classes of glia cells in the optic lobes of Drosophila melanogaster were defined by the enhancer trap technique, using expression of the lacZ reporter gene. At both the outer and inner optic chiasms, there are stacks of glia, arrayed from dorsal to ventral, interpersed between the crossings of axonal fiber bundles. The giant glial cells of both the outer and inner chiasms are similar with respect to their nuclear shapes and positions, indicating similar functions of these cell types. Another class of glia is found in the medulla neuropil. Their cell bodies anchor in the most distal region of the neuropil, and their processes extend into the deeper neuropil layers. Birth dating using BrdU shows that both groups of chiasm glia are born early in larval life; they may participate in the development of the optic lobe. The medulla glia are born later and may be involved primarily in adult functions. In the wild type, and in mutants with structurally altered optic lobes, the numbers of tract-associated glial cells in the outer and inner optic chiasms seem to vary with the number of visual columns, whereas the complement of medulla neuropil glia correlates with the volume of the optic lobe.

The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila.

Swiss cheese (sws) mutant flies develop normally during larval life but show age-dependent neurodegeneration in the pupa and adult and have reduced life span. In late pupae, glial processes form abnormal, multilayered wrappings around neurons and axons. Degeneration first becomes evident in young flies as apoptosis in single scattered cells in the CNS, but later it becomes severe and widespread. In the adult, the number of glial wrappings increases with age. The sws gene is expressed in neurons in the brain cortex. The conceptual 1425 amino acid protein shows two domains with homology to the regulatory subunits of protein kinase A and to conceptual proteins of yet unknown function in yeast, worm, and human. Sequencing of two sws alleles shows amino acid substitutions in these two conserved domains. It is suggested that the novel SWS protein plays a role in a signaling mechanism between neurons and glia that regulates glial wrapping during development of the adult brain.

Calx, a Na-Ca exchanger gene of Drosophila melanogaster.

We have cloned Calx, a gene that encodes a Na-Ca exchanger of Drosophila melanogaster. Calx encodes two repeated motifs, Calx-alpha and Calx-beta, that overlap domains required for exchanger activity and regulation. Calx has multiple transcripts in adults, including at least one expressed in the retina. The Calx genomic locus comprises >/=35 kb between the Atpalpha and rudimentary-like genes in chromosomal region 93B. In Xenopus oocytes, microinjected Calx cRNA induces calcium uptake like that of its homolog, the 3Na+-1Ca2+ exchanger of mammalian heart. Implications of Calx-alpha motifs for the mechanism of Na-Ca exchange are discussed.

Drosophila drop-dead mutations accelerate the time course of age-related markers.

Mutations of the drop-dead gene in Drosophila melanogaster lead to striking early death of the adult animal. At different times, after emergence from the pupa, individual flies begin to stagger and, shortly thereafter, die. Anatomical examination reveals gross neuropathological lesions in the brain. The life span of flies mutant for the drop-dead gene is four to five times shorter than for normal adults. That raises the question whether loss of the normal gene product might set into motion a series of events typical of the normal aging process. We used molecular markers, the expression patterns of which, in normal flies, change with age in a manner that correlates with life span. In the drop-dead mutant, there is an acceleration in the temporal pattern of expression of these age-related markers.

Generation of cDNA expression libraries enriched for in-frame sequences.

Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

Spongecake and eggroll: two hereditary diseases in Drosophila resemble patterns of human brain degeneration.

Various neuronal degenerative diseases are characterized by late onset, relentless progression, and finally death. Many have a direct genetic basis; others are of still unknown etiological mechanisms [1,2]. The study of human neurodegenerative diseases is complicated by the difficulty of obtaining tissue samples at various stages of progression, especially early in the course of the disease. Since neurodegeneration occurs in many organisms [3-5], model organisms amenable to genetic and molecular techniques, such as the mouse, offer important advantages. Much less laborious and expensive are worms or flies, which have short generation times and can be rapidly screened for mutations. To investigate the use of the fly as a model system for identifying genes related to such diseases, we screened for mutants having reduced lifespan, then examined them for brain degeneration. We describe here two such mutants, each with a different pattern of degeneration as characterized by light and transmission electron microscopy. The brain of the aging spongecake mutant exhibits regionally specific, membrane-bound vacuoles similar to those seen in spongiform degenerations such as Creutzfeldt-Jakob disease [6,7]. The mutant eggroll develops dense, multilamellated structures in the brain, resembling ones found in lipid storage diseases such as Tay-Sachs [8].

Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+.

The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1-10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.

Independent determination of symmetry and polarity in the Drosophila eye.

In each facet of the Drosophila compound eye, a cluster of photoreceptor cells assumes an asymmetric trapezoidal pattern. These clusters have opposite orientations above and below an equator, showing global dorsoventral mirror symmetry. However, in the mutant spiny legs, the polarization of each cluster appears to be random, so that no equator is evident. The apparent lack of an equator suggests that spiny legs+ may be involved in the establishment of global dorsoventral identity that might be essential for proper polarization of the photoreceptor clusters. Alternatively, a global dorsoventral pattern could be present, but spiny legs+ may be required for local polarization of individual clusters. Using an enhancer trap strain in which white+ gene expression is restricted to the dorsal field, we show that white+ expression in spiny legs correctly respects dorsoventral position even in facets with inappropriate polarizations; the dorsoventral boundary is indeed present, whereas the mechanism for polarization is perturbed. It is suggested that the boundary is established before the action of spiny legs+ by an independent mechanism.

Behavioral genetics of thermosensation and hygrosensation in Drosophila.

Whereas temperature and humidity are critical variables affecting physiology, behavior, and evolution, the genetic and neuronal underpinnings of thermosensation and hygrosensation remain poorly understood. We have initiated a behavioral-genetic investigation of these sensory systems in Drosophila. Behavioral tests are described for the rapid screening of mutants defective in thermosensation and hygrosensation. We demonstrate the strong responses of normal flies to temperature and humidity. Two mutants were found with defects in thermosensation, only one of which is also defective in hygrosensation, indicating that they involve different sensory mechanisms. Ablation experiments further separate these sensory systems by showing that thermoreceptors are housed in the third antennal segment, whereas hygroreceptors are located more distally in the antennal arista.

Transvection at the eyes absent gene of Drosophila.

The Drosophila eyes absent (eya) gene is required for survival and differentiation of eye progenitor cells. Loss of gene function in the eye results in reduction or absence of the adult compound eye. Certain combinations of eya alleles undergo partial complementation, with dramatic restoration of eye size. This interaction is sensitive to the relative positions of the two alleles in the genome; rearrangements predicted to disrupt pairing of chromosomal homologs in the eya region disrupt complementation. Ten X-ray-induced rearrangements that suppress the interaction obey the same general rules as those that disrupt transvection at the bithorax complex and the decapentaplegic gene. Moreover, like transvection in those cases, the interaction at eya depends on the presence of normal zeste function. The discovery of transvection at eya suggests that transvection interactions of this type may be more prevalent than generally thought.