Variance Component Analysis of Allozyme Frequency Data from Eastern Populations of DROSOPHILA MELANOGASTER

Gene frequency variation at eight polymorphic allozyme loci in Drosophila melanogaster populations in North Carollina and the east coast of the United States were analyzed utilizing the variance component estimation procedures suggested by Cockerham (1969, 1973). These variance components were used to estimate correlations of genes within small geographic regions. The average (over loci) correlation between genes in the same individual within subpopulations was estimated to be 0.033. That between genes in the same subpopulation in different individuals was estimated to be very small, although significantly different from zero. The macrogeographic variation measured by the correlation of genes sampled from the same local region was large for some loci and smaller for others. this variation was also analyzed by correlation with latitude and longitude. Several previously recognized clines were identified as were several new clines.--These results were interpreted as indicating either some degree of nonrandom mating and local breeding unit isolation or a low frequency of null alleles. The geographic and temporal variation has no simple interpretation.     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Optimal conditions for studies of amino acid incorporation in vitro by isolated skeletal muscle mitochondria

Optimal conditions for amino acid incorporation into protein in vitro by isolated skeletal muscle mitochondria were established. Maximum incorporation rates were obtained when atractylate and glutamate were added to the incubation medium in the absence of any exogenous adenine nucleotides. Under these conditions, the rate of amino acid incorporation was more than 5-fold greater than that observed with glutamate and ADP and nearly 12-fold greater than that observed with ATP and an ATP-regenerating system consisting of phosphoenolpyruvate and pyruvate kinase. The optimal concentrations of adenine nucleotides, glutamate, cofactors and the substrate leucine were determined for all three energy-providing systems. The inhibitors of protein synthesis, puromycin and chloramphenicol, completely blocked amino acid incorporation by isolated skeletal muscle in mitochondria, while cycloheximide had no effect. Analysis of the labeled mitochondrial proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis revealed five labeled bands of molecular weights ranging from 38,000 to 10,000.Amino acid incorporation by skeletal muscle mitochondria isolated from diabetic rats was decreased over 60% as compared to mitochondria from controls when measured in the presence of glutamate and atractylate, ADP and glutamate or the ATP regenerating system. By contrast, amino acid incorporation by liver mitochondria isolated from diabetic rats did not differ significantly from control values when measured with four different energy sources.     

The antagonism by anti-inflammatory analgesics of prostaglandin F2α-induced contractions of human and rabbit myometrium

The anti-inflammatory analgesic drugs, aspirin, indomethacin, phenylbutazone, mefenamic acid, ibuprofen and flurbiprofen are shown to inhibit in a dose-dependent manner the force of contraction of isolated human pregnant myometrial strips which have been stimulated to contract by adding prostaglandin (PG) F_2α to the tissue bath. These drugs and also flufenamic acid and salicin show a similar antagonism of the action of PGF_2α with isolated rabbit non-pregnant myometrium. The ratio of the inhibitory concentration to the maximum plasma level after a normal dose suggests that phenylbutazone and possibly ibuprofen may be capable of inhibiting human uterine contractions . Patients who were treated with aspirin during induction of abortion using PGF_2α during the second trimester of pregnancy showed no significant change in the induction-abortion interval compared with patients not taking aspirin.     

Frequent Unanticipated Alleles of lethal giant larvae in Drosophila Second Chromosome Stocks

Forty years ago, a high frequency of lethal giant larvae (lgl) alleles in wild populations of Drosophila melanogaster was reported. This locus has been intensively studied for its roles in epithelial polarity, asymmetric neural divisions, and restriction of tissue proliferation. Here, we identify a high frequency of lgl alleles in the Bloomington second chromosome deficiency kit and the University of California at Los Angeles Bruinfly FRT40A-lethal P collection. These unrecognized aberrations confound the use of these workhorse collections for phenotypic screening or genetic mapping. In addition, we determined that independent alleles of insensitive, reported to affect asymmetric cell divisions during sensory organ development, carry lgl deletions that are responsible for the observed phenotypes. Taken together, these results encourage the routine testing of second chromosome stocks for second-site alleles of lgl.     

Gain and loss of fluid in metamorphosing larvae of Manduca sexta

Larvae of Manduca sexta, (Sphingidae) increase their weight approx. 50% just before pupation and then secrete this fluid during the formation of their burrows. Time-lapse cinematography indicates that the fluid is ejected into the walls of the final burrow. It may offer some mechanical support; it is not particularly repellent to ants or mice, and it contains only small amounts of the alkaloids ingested from its preferred food plants. Comparison to other species indicates that the gain and loss of water is associated with burrowing behaviour; the fluid appears to be used in providing hydraulic pressure for burrowing, in forming and cementing the pupal chamber, and in acting as a CO₂ trap underground. The secretion is a hypertonic, highly alkaline solution containing KHCO₃ and small amounts of Na⁺, Ca²⁺, Mg²⁺, PO₄^?3 and some proteins. Haemolymph levels of K⁺ decrease, and those of Ca²⁺ increase, during the secretory phase. When radioactive calcium is injected into mature larvae, it appears promptly in the secretion. If however, the injection is given more than 24 hr before the animal begins secreting, then the calcium is bound to haemolymph protein and does not appear in the secretion.     

Sequence- and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine.

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.     

A Chemical Proteomics Approach for the Search of Pharmacological Targets of the Antimalarial Clinical Candidate Albitiazolium in Plasmodium falciparum Using Photocrosslinking and Click Chemistry

Plasmodium falciparum is responsible for severe malaria which is one of the most prevalent and deadly infectious diseases in the world. The antimalarial therapeutic arsenal is hampered by the onset of resistance to all known pharmacological classes of compounds, so new drugs with novel mechanisms of action are critically needed. Albitiazolium is a clinical antimalarial candidate from a series of choline analogs designed to inhibit plasmodial phospholipid metabolism. Here we developed an original chemical proteomic approach to identify parasite proteins targeted by albitiazolium during their native interaction in living parasites. We designed a bifunctional albitiazolium-derived compound (photoactivable and clickable) to covalently crosslink drug–interacting parasite proteins in situ followed by their isolation via click chemistry reactions. Mass spectrometry analysis of drug–interacting proteins and subsequent clustering on gene ontology terms revealed parasite proteins involved in lipid metabolic activities and, interestingly, also in lipid binding, transport, and vesicular transport functions. In accordance with this, the albitiazolium-derivative was localized in the endoplasmic reticulum and trans-Golgi network of P. falciparum. Importantly, during competitive assays with albitiazolium, the binding of choline/ethanolamine phosphotransferase (the enzyme involved in the last step of phosphatidylcholine synthesis) was substantially displaced, thus confirming the efficiency of this strategy for searching albitiazolium targets.