Direct purification of polyadenylated RNAs from isolated polysome fractions

We report a simplified and improved method for obtaining polyadenylated RNAs (poly(A) RNAs) from polysome fractions. Isolated polysomes were subjected directly to poly(U)-Sephadex column chromatography without conventional purification of polysomal RNAs by phenol extraction followed by ethanol precipitation. The yield of poly(A) RNAs by this direct purification method was about twice that obtained by the conventional method. When the two poly(A) RNA preparations were used in two-dimensional polyacrylamide gel electrophoretic analysis of cell-free translation products and cDNA synthesis, biological activity of the directly purified poly(A) RNA was equal to or even better than that of conventionally purified poly(A) RNA.     

Runaway evolution of the male-specific exon of the doublesex gene in Diptera

In Diptera (Insecta), alternatively spliced male-specific and female-specific products of the doublesex (dsx) gene play a key role in regulating development of the adult genital structures from the genital disc. Analysis of the pattern of nucleotide substitution of different domains of the dsx gene in 29 dipteran species showed that, over short evolutionary times, purifying selection predominated on the domain common to both sexes, the female-specific exons, and the and male-specific exon. However, over longer the evolutionary time frames represented by between-family comparisons, the male-specific exon accumulated nonsynonymous substitutions at a much more rapid rate than either the common domain or the female-specific exon. Overall, the accumulation of nonsynonymous substitutions in the male-specific exon occurred at a significantly greater than linear rate relative to the common domain, whereas the accumulation of nonsynonymous substitutions in the female-specific exon occurred at less than linear rate relative to the common domain. The evolution of the male-specific exon of dsx thus shows a pattern reminiscent of that seen in the "runaway" evolution of male secondary sexual characters at the morphological level, consistent with the hypothesis that female choice is an important factor in the morphological diversification of insect male genitalia.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

The expression of a plant genome in hnRNA and mRNA.

Representation of genomic kinetic sequence classes and sequence complexities were investigated in nuclear and polysomal RNA of the higher plant Petroselinum sativum (parsley). Two different methods indicated that most if not all polysomal poly(A) -RNA is transcribed from unique sequences. As measured by saturation hybridization in root callus and young leaves 8.7% and 6.2%, respectively, of unique DNA were transcribed in mRNA corresponding to 13.700 and 10.000 average sized genes. Unique nuclear DNA hybridized with an excess of polysomal poly(A)mRNA to the same extent as with total polysomal RNA. 3H-cDNA - poly(A)mRNA hybridization kinetics revealed the presence of two abundance classes with 9.200 and about 30 different mRNAs in leaves and two abundance classes with 10.500 and 960 different mRNAs in callus cells. The existence of plant poly(A)hnRNA was proven both by its fast kinetics of appearance, its length distribution larger than mRNA, and its sequence complexity a few times that of polysomal RNA.     

Detection of quantitative trait loci for body weight at 10 weeks from Philippine wild mice

A genome-wide scan for quantitative trait loci (QTLs) controlling body weight at 10 weeks after birth was carried out in a population of 387 intersubspecific backcross mice derived from a cross between C57BL/6J inbred mice (Mus musculus domesticus) and wild mice (M. m. castaneus) captured in the Philippines, in order to discover novel QTLs from the wild mice that have about 60% lower body weight than C57BL/6J. By interval mapping, we detected four QTLs: a highly significant QTL on Chromosome (Chr) 2, which was common in both sexes; two significant QTLs on Chr 13, one male-specific and the other female-specific; and a suggestive male-specific QTL on X Chr. By composite interval mapping, we confirmed the presence of the three QTLs on Chrs 2 and 13, but not of the male-specific X-linked QTL. The composite interval mapping analysis newly identified three QTLs: a significant male-specific QTL on Chr 11 and two highly significant female-specific QTLs on Chrs 9 and X. Individual QTLs explained 3.8–11.6% of the phenotypic variance, and all the QTL alleles derived from the wild mice decreased body weight. A two-way analysis of variance revealed a significant epistatic interaction between the Chr 2 QTL and the background marker locus D12Mit4 on Chr 12 only in males. The interaction effect unexpectedly increased body weight. The chromosomal region containing the Chr 2 QTL did not coincide with those of growth or fatness QTLs mapped in previous studies. These results suggest that a population of wild mice may play an important role as new sources of valuable QTLs. Received: 14 January 2000 / Accepted: 14 April 2000     

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.     

Ectopic expression of the female transformer gene product leads to female differentiation of chromosomally male Drosophila

The transformer (tra) gene of Drosophila is necessary for all aspects of female somatic sexual differentiation. tra uses a single set of precursor RNAs to produce female- and non-sex-specific RNAs by alternative splicing. Ectopic expression of the female-specific RNA causes chromosomal males to develop as females, indicative of a linear pathway of regulated genes controlling sex. Genetic and molecular tests with this ectopically expressed gene are consistent with the following order of gene action: X chromosome to autosome ratio----Sex lethal----transformer----transformer-2----doublesex----intersex--- - terminal differentiation. Expression of the female-specific tra RNA in tra mutants is sufficient to lead to female differentiation. Expression of the non-sex-specific tra RNA in tra mutants is not sufficient to lead to female differentiation. The tra female-specific activity is not required for female-specific splicing of the tra precursor RNAs.     

Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of delta 4-steroid 5 alpha-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450

Neonatal gonadectomy studies and hormonal replacement regimens were employed to characterize the regulation of delta 4-steroid 5 alpha-reductase, microsomal flavin-containing monooxygenase, and several forms of rat hepatic microsomal cytochrome P-450, including three that are sexually differentiated. Rats of both sexes that had been gonadectomized at birth were either untreated or were administered testosterone propionate or estradiol benzoate neonatally (subcutaneous injection on days 1 and 3 of life), postpubertally (an implant of a hormone-packed capsule at 5 weeks of age), or both neonatally and postpubertally. At the age of 10 weeks, all rats were killed, and several liver microsomal enzymes were assayed using immunochemical and catalytic techniques. Expression in the 10-week-old male and female rats of two male-specific cytochrome P-450 forms, termed P-4502c/UT-A and P-4502a/PCN-E, and their associated respective 16 alpha- and 6 beta-steroid hydroxylase activities could either be imprinted (programmed) by androgen exposure during the early neonatal period or, alternatively, could be stimulated by continuous hormone treatment after the age of 5 weeks. By contrast, hepatic expression of two female-specific enzymes, P-4502d/UT-1 and delta 4-steroid 5 alpha-reductase, was only partially dependent on estradiol; birth-gonadectomized rats expressed as much as 30-50% of the enzyme levels present in untreated adult females. Expression of both female-specific enzymes was fully suppressed upon postpubertal exposure to testosterone. In another study, birth sham-operated female rats were administered testosterone using the same regimens described above for the birth-gonadectomized rats. Although neonatal testosterone treatment alone did not affect the expression in these females of the four sex-specific enzymes examined in this study, it did enhance significantly the masculinization effected by postpubertal androgen exposure. This resulted in expression of the male-specific enzymes P-4502c/UT-A and P-4502a/PCN-E in these females at levels comparable to those found in adult males, while simultaneously suppressing the two female-specific enzymes, P-4502d/UT-I and delta 4-steroid 5 alpha-reductase, by approximately 70-75% to levels characteristic of prepubertal rats of either sex. The levels of another microsomal enzyme, flavin-containing monooxygenase, were also measured and found to be regulated by testosterone, but the ontogenic profiles and the effects of gonadectomy and hormone replacement indicated clear differences in its regulation when compared to the other male-specific enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

mRNA usage during Drosophila melanogaster embryonic development. Analysis of nine cloned DNA segments

A set of nine phage lambda clones containing inserts from Drosophila melanogaster which are complementary to cDNA made from oocyte poly(A)+ RNA were selected from a larger group. These cloned elements code for a range of middle abundant RNA sequences which show no appreciable change in abundance during Drosophila embryogenesis. Seven of the nine clones are complementary to two oocyte RNAs, one to three RNAs and one to four RNAs. This study describes the changes that occur in these RNAs during embryonic development in the polysomal and non-polysomal fraction, and in the poly(A)+ RNA and poly(A)- RNA fraction. In all nine of these clones, greater than 70% of the complementary RNA is found in the polysomal region of a sucrose gradient. This proportion increases somewhat during development. Specific changes have been found during development in the proportion of RNA that is poly(A)+. Depending to the cloned sequence, this proportion may increase, decrease, or remain unchanged. For those clones that show a change, most of this change occurs between 8 and 19 h of development. Our data suggest, furthermore, the presence of a class of non-adenylated RNA being utilized during embryogenesis.     

An electrophoretic study of proteins of the ovary,fat body,and haemolymph in the migratory grasshopper, Melanoplus sanguinipes

Electrophoretic separation of saline extracts from the ovary revealed 14 proteins. Twelve proteins were detected in the fat body, of which seven had electrophoretic mobilities identical to those in the ovary. Similarly, eight of 16 proteins in the haemolymph of vitellogenic females ahad electrophoretically identical counterparts in the ovary. As these proteins accumulate in the haemolymph of ovariectomized females, the findings suggest that most yolk proteins are synthesized in the fat body. Although most female haemolymph proteins are present in males, two of the predominant yolk protiens are absent and represent female-specific proteins.Although certain proteins accumulate in the haemolymph of allatectomized females, the major ovarian proteins are absent or present in low concentrations. However, 48 hr after allatectomized females are treated with a juvenile hormone analogue, the haemolymph protein pattern resembles that of a normal female. This suggests that the corpora allata stimulate the synthesis of female-specific and other vitellogenic proteins. The median neurosecretory cells (mNSC) are also necessary for synthesis of female-specific proteins. Furthermore, proteins which are present in allatectomized females are absent in mNSC-cauterized insects suggesting that the mNSC stimulate general protein synthesis.     

Identification and characterization of sex-linked proteins of Schistosoma mansoni.

The proteins of adults worms (male and female) of two isolates (BH and RJ) of Schistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of male and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 KDa, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females proteins were detected by Western blotting using a sera from infected Nectomys squamipes.     

Identification of a male-specific AFLP marker in a functionally dioecious fig,<Emphasis Type="Italic"> Ficus fulva</Emphasis> Reinw. ex Bl. (Moraceae)

A male-specific amplified fragment length polymorphism (AFLP) marker was identified in the functionally dioecious fig species, Ficus fulva. A total of 89 polymorphic fragments from three primer combinations were produced, of which one (246 bp) was present in all males (n=23) and absent in all females (n=24) of two populations. This strong association suggests a tight chromosomal linkage between the AFLP marker and the sex-controlling locus. Further analysis indicated that the marker segregated in open-pollinated progenies from natural populations in a 1:1 ratio (n=156), implying that males are the heterogametic sex. Chromosome preparations showed no evidence for morphologically distinct sex chromosomes. The low frequencies of associated markers argue against a morphologically cryptic non-recombining sex chromosome. The sex-locus is therefore likely to be autosomal. The male-specific AFLP marker was sequenced and converted into a sequence characterised amplified region (SCAR) marker. This SCAR marker produced a fragment of equal size in males and females, suggesting that sequence divergence between male- and female-specific chromosomal regions is low.Publication 3311 NIOO-KNAW Netherlands Institute of Ecology     

Growth hormone pretranslationally regulates the sexually dimorphic expression of the prolactin receptor gene in rat liver

The female-specific expression of the rat liver PRL receptor (PRL-R) gene was investigated by Northern analysis of hypophysectomized rats after two alternative human GH treatments that were to mimic either 1) the continuous female-specific or 2) the discontinuous male-specific serum GH patterns. The former (female-specific) pattern was shown to result in a dramatic increase in PRL-R mRNA in both males and females, while the latter (male-specific) pattern failed to evoke this response. A similar inductive effect in hypophysectomized females was shown after continuous administration of bovine GH and was found to constitute an approximately 60-fold increase in PRL-R mRNA levels. This effect by bovine GH, which, unlike the human isoform, is devoid of lactogenic properties, thus indicates the somatogenic origin of the signal resulting in this inductive response. These observations in conjunction with previous data obtained for other GH-regulated nonreceptor genes are interpreted to support the proposal of GH serum patterns being an early signal in a more general mechanism for pretranslational regulation of sex-specific gene expression. In contrast to GH, only a slight elevation of PRL-R mRNA was evoked by the ligand ovine PRL, while coadministration of ovine PRL with bovine GH failed to enhance the mRNA level found with bovine GH alone. The detection of previously unreported PRL-R mRNAs in liver of approximately 3.0, 3.8, and 5 kilobases in addition to the major 2.2-kilobase form was also evident after continuous GH administration.