Isolation and characterization of perithecial development mutants in neurospora

The isolation and characterization of mutants that block perithecial development in Neurospora crassa are described. Several classes of mutants have been isolated after UV mutagenesis, and those that block perithecial development when used as the female (protoperithecial) component of a cross have been further characterized. These mutants fall into 29 complementation groups. Twelve of the 33 mutants block development at the protoperithecial stage; no other clustering of block points is observed. Many of the mutants show an altered vegetative growth rate as well; in several mutants this lower growth rate cosegregates with the female sterile phenotype. Only one mutant also blocks development of the perithecium when used as the conidial parent. None of the mutants are temperature sensitive; two can be suppressed by growth on a complete crossing medium. There is no indication that the mutants are at or in the mating-type locus, nor are any of the mutants mating-type specific. Genetic mosaics have been formed using mixtures of mutant and marked wild-type nuclei; no mutants are cell autonomous by this criterion. The significance of these results in terms of "developmental" mutants isolated in other organisms and in relation to models of eukaryotic development is discussed.     

Analysis of male pheromones that accelerate female reproductive organ development

Male odors can influence a female's reproductive physiology. In the mouse, the odor of male urine results in an early onset of female puberty. Several volatile and protein pheromones have previously been reported to each account for this bioactivity. Here we bioassay inbred BALB/cJ females to study pheromone-accelerated uterine growth, a developmental hallmark of puberty. We evaluate the response of wild-type and mutant mice lacking a specialized sensory transduction channel, TrpC2, and find TrpC2 function to be necessary for pheromone-mediated uterine growth. We analyze the relative effectiveness of pheromones previously identified to accelerate puberty through direct bioassay and find none to significantly accelerate uterine growth in BALB/cJ females. Complementary to this analysis, we have devised a strategy of partial purification of the uterine growth bioactivity from male urine and applied it to purify bioactivity from three different laboratory strains. The biochemical characteristics of the active fraction of all three strains are inconsistent with that of previously known pheromones. When directly analyzed, we are unable to detect previously known pheromones in urine fractions that generate uterine growth. Our analysis indicates that pheromones emitted by males to advance female puberty remain to be identified.     

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.     

Embryonic expression of the common progeroid lamin A splice mutation arrests postnatal skin development

Hutchinson–Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) are two laminopathies caused by mutations leading to cellular accumulation of prelamin A or one of its truncated forms, progerin. One proposed mechanism for the more severe symptoms in patients with RD compared with HGPS is that higher levels of farnesylated lamin A are produced in RD. Here, we show evidence in support of that hypothesis. Overexpression of the most common progeroid lamin A mutation (LMNA c.1824C>T, p.G608G) during skin development results in a severe phenotype, characterized by dry scaly skin. At postnatal day 5 (PD5), progeroid animals showed a hyperplastic epidermis, disorganized sebaceous glands and an acute inflammatory dermal response, also involving the hypodermal fat layer. PD5 animals also showed an upregulation of multiple inflammatory response genes and an activated NF‐kB target pathway. Careful analysis of the interfollicular epidermis showed aberrant expression of the lamin B receptor (LBR) in the suprabasal layer. Prolonged expression of LBR, in 14.06% of the cells, likely contributes to the observed arrest of skin development, clearly evident at PD4 when the skin had developed into single‐layer epithelium in the wild‐type animals while progeroid animals still had the multilayered appearance typical for skin at PD3. Suprabasal cells expressing LBR showed altered DNA distribution, suggesting the induction of gene expression changes. Despite the formation of a functional epidermal barrier and proven functionality of the gap junctions, progeroid animals displayed a greater rate of water loss as compared with wild‐type littermates and died within the first two postnatal weeks.     

Wnt4 is required for proper male as well as female sexual development

Genes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad and we showed previously that the role of WNT4 was to inhibit endothelial and steroidogenic cell migration into the developing ovary. Here we show that Wnt4 also has a function in the male gonad. We find that Sertoli cell differentiation is compromised in Wnt4 mutant testes and that this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 -/- gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development.     

A dominant allele controls development into female mimic male and diminutive female ruffs

Maintaining polymorphisms for genes with effects of ecological significance may involve conflicting selection in males and females. We present data from a captive population of ruffs (Philomachus pugnax) showing that a dominant allele controls development into both small, ‘female mimic’ males (‘faeders’), and a previously undescribed class of small ‘female faeders’. Most male ruffs have elaborate breeding plumage and display behaviour, but 0.5–1.5% are faeders, which lack both. Females from a captive population previously lacking faeders were bred with two founder faeder males and their faeder sons. The faeders’ offspring had a quadrimodal size distribution comprising normal-sized males and females, faeders and atypically small females. By contrast, ornamented males fathered only normal-sized offspring. We conclude that both founding faeders were heterozygous for a faeder allele absent from the original population. This allele is dominant to previously described genes that determine development into independent versus satellite ornamented males. Unlike those genes, the faeder allele is clearly expressed in females. Small body size is a component of the male faeder mating strategy, but provides no obvious benefit to females. Bisexual expression of the gene provides the opportunity to quantify the strength of sexually antagonistic selection on a Mendelian trait.     

A meiotic uv-sensitive mutant that causes deletion of duplications in neurospora

The meiotic-3 (mei-3) mutant of Neurospora crassa has several effects: (1) When homozygous, it almost completely blocks meiosis and ascospore formation, (2) it is sensitive to UV, (3) its growth is inhibited by histidine and, (4) it increases the instability of nontandem duplications. This was shown for duplications produced by five different rearrangements and was demonstrated by two different criteria. The effects on meiosis and duplication instability are expressed strongly at 25°; the effects on sensitivity to UV and to histidine are expressed strongly at 38.5° but only slightly at 25°. Nevertheless, all four effects were shown to be due to a single gene. mei-3 is not allelic with previously reported UV-sensitive mutants.—Two other results were obtained that are not necessarily due to mei-3: (1) A cross involving mei-3 produced a new unlinked meiotic mutant, mei-4, which is not sensitive to UV or histidine, and (2) a burst of several new mutants occurred in a different mei-3 stock, including a partial revertant of mei-3.—mei-3 has previously been shown to cause frequent complete loss of a terminal duplicate segment, beginning exactly at the original rearrangement breakpoint. Possible mechanisms are discussed by which a UV-sensitive mutant could cause such precise deletions.     

Nafoxidine administered to newborn female GR mice arrests the development of their mammary glands

Mice of the GR strain develop many hormone-dependent mammary tumors in response to estrogen and progesterone stimulation. Since this strain is so sensitive to steroid hormones, we administered a single dose of the antiestrogen Nafoxidine to female GR mice within 24 hours after their birth. This treatment arrested the development of their mammary glands and when the mice were adults, 10 weeks old, they did not cycle normally but were in a state of persistent estrus. Whole mounts of mammary glands from Nafoxidine-treated mice revealed cystic areas within some ducts and bulbous swellings at the ends of others. No hyperplastic alveolar nodules (HAN) were identified in the glands. In contrast, a single dose of 17 beta estradiol administered within 24 h after birth, resulted in a highly branched gland displaying typical end buds, a few alveoli and more HAN than were observed in glands of control adult mice of the same strain. Thus Nafoxidine treatment not only arrested the development of the mammary glands in female GR mice (causing them to appear "masculinized") but it also produced abnormalities within the glands.     

海桐大、小孢子发育及雌、雄配子体形成的研究

利用常规石蜡制片法研究了海桐大、小孢子发生及雌、雄配子体发育的过程。结果显示:(1)小孢子母细胞减数分裂过程中的胞质分裂为连续型,四分孢子为以四面体形为主,四分孢子后期部分小孢子壁皱缩;(2)花药壁由4层结构组成,由外到内为表皮、药室内壁、中层和绒毡层;(3)海桐具多个胚珠,单珠被,薄珠心,胚珠类型为倒生胚珠。大孢子母细胞减数分裂主要形成线形排列的4个大孢子,还具有少有的十字形排列,功能大孢子位于合点端;(4)胚囊发育属单孢型的蓼型,成熟的雌配子体为四细胞五核胚囊。     

Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.     

A mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium

The expression of an activated RasG, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild-type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (M. Khosla et al., 1996, Mol. Cell. Biol. 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG-G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signaling through the effector domain was required for both the morphological and the development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effector.     

A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster

The adult male accessory glands of D. melanogaster synthesize and secrete a peptide that represses female sexual receptivity and stimulates oviposition. Normally, this peptide is transferred to females during copulation; however, the peptide shows the same biological activity after purification and subsequent injection into the abdominal cavity of female virgins. Amino acid sequencing of the purified peptide and oligonucleotide-directed cDNA cloning established that the peptide consists of 36 amino acids. It appears to be synthesized as a precursor with a hydrophobic signal sequence of 19 residues at its N-terminal end. The precursor peptide is encoded by a short mRNA that accumulates exclusively in the male accessory gland. The gene has been localized by in situ hybridization to polytene chromosomes at 70A.     

A cation-pi interaction discriminates among sodium channels that are either sensitive or resistant to tetrodotoxin block

Voltage-gated sodium channels control the upstroke of the action potential in excitable cells of nerve and muscle tissue, making them ideal targets for exogenous toxins that aim to squelch electrical excitability. One such toxin, tetrodotoxin (TTX), blocks sodium channels with nanomolar affinity only when an aromatic Phe or Tyr residue is present at a specific location in the external vestibule of the ion-conducting pore. To test whether TTX is attracted to Tyr401 of NaV1.4 through a cation-pi interaction, this aromatic residue was replaced with fluorinated derivatives of Phe using in vivo nonsense suppression. Consistent with a cation-pi interaction, increased fluorination of Phe401, which reduces the negative electrostatic potential on the aromatic face, caused a monotonic increase in the inhibitory constant for block. Trifluorination of the aromatic ring decreased TTX affinity by approximately 50-fold, a reduction similar to that caused by replacement with the comparably hydrophobic residue Leu. Furthermore, we show that an energetically equivalent cation-pi interaction underlies both use-dependent and tonic block by TTX. Our results are supported by high level ab initio quantum mechanical calculations applied to a model of TTX binding to benzene. Our analysis suggests that the aromatic side chain faces the permeation pathway where it orients TTX optimally and interacts with permeant ions. These results are the first of their kind to show the incorporation of unnatural amino acids into a voltage-gated sodium channel and demonstrate that a cation-pi interaction is responsible for the obligate nature of an aromatic at this position in TTX-sensitive sodium channels.     

Evidence that genes from the male parent may influence the morphology of potato dihaploids

A number of recent studies have provided evidence that potato dihaploids (S. tuberosum) contain and express DNA from the male (dihaploid inducer) parent, S. phureja. The importance of this for breeding programmes that use dihaploid potatoes is to some extent dependent upon whether the S. phureja DNA influences dihaploid morphology. In the present study, 21 characters were used to compare the morphology of six dihaploids with those of their parents: S. tuberosum (cvs `Pentland Dell' and `Pentland Crown') and S. phureja (IVP48). Characteristics of S. phureja were found in all of the dihaploids examined. In principal component analyses, dihaploids formed intermediate groupings positioned between those of the parents, although much closer to S. tuberosum. It is concluded there is evidence that DNA originating from the dihaploid inducer can affect the morphology of potato dihaploids. Implications of the findings are discussed. Received: 26 November 1995 / Accepted: 9 February 1996     

Bio-soliton model that predicts non-thermal electromagnetic frequency bands,that either stabilize or destabilize living cells

Solitons, as self-reinforcing solitary waves, interact with complex biological phenomena such as cellular self-organization. A soliton model is able to describe a spectrum of electromagnetism modalities that can be applied to understand the physical principles of biological effects in living cells, as caused by endogenous and exogenous electromagnetic fields and is compatible with quantum coherence. A bio-soliton model is proposed, that enables to predict which eigen-frequencies of non-thermal electromagnetic waves are life-sustaining and which are, in contrast, detrimental for living cells. The particular effects are exerted by a range of electromagnetic wave eigen-frequencies of one-tenth of a Hertz till Peta Hertz that show a pattern of 12 bands, and can be positioned on an acoustic reference frequency scale. The model was substantiated by a meta-analysis of 240 published articles of biological electromagnetic experiments, in which a spectrum of non-thermal electromagnetic waves were exposed to living cells and intact organisms. These data support the concept of coherent quantized electromagnetic states in living organisms and the theories of Fröhlich, Davydov and Pang. It is envisioned that a rational control of shape by soliton-waves and related to a morphogenetic field and parametric resonance provides positional information and cues to regulate organism-wide systems properties like anatomy, control of reproduction and repair.     

Wing development in parasitized male and female Sitobion fragariae

Abstract.Different stages of presumptive winged morphs (males, gynoparae and alate virginoparae) of the blackberry‐cereal aphid, Sitobion fragariae , were exposed to attack by Aphidius ervi . Even though the mechanism influencing wing development in the three aphid morphs differs, the effects of parasitism were similar. Alatiform structures were completely inhibited in all three morphs when the initial attack took place in their first or early second stadium. The disruption of wing development also resulted in apterous/alate‐intermediate forms when aphids were attacked from first (males and gynoparae) or early second (alate virginoparae) up to the fourth larval stadium. The fact that wing development was still disrupted when aphids with well developed wingbuds were parasitized indicates that the early stages of parasitization were influential. Thus, the morphogenetic effects may be exerted by the parasitoid egg or calyx fluid.     

A new study challenges the current belief of a high human male:female mutation ratio

A recent comparison of a DNA region that was transposed from the X to the Y chromosome 3–4 million years ago, with the same region on the X chromosome showed only a slight excess of mutant changes on the Y chromosome. This translates to an estimate of 1.7 for the ratio of the male to female mutation rate, much less than the average 5.1 of previous studies. The authors argue that this throws doubt not only on higher male mutation rates in human ancestry, but also on the standard assumption of a high male:female ratio in contemporary human populations. Clearly, more studies are needed to clear up this discrepancy in the ancestral rates, but I believe that the high contemporary male:female ratio for base substitutions is too well established to be overthrown by even a very good evolutionary study.