Population Genetics of Tandem Repeats in Centromeric Heterochromatin: Unequal Crossing over and Chromosomal Divergence at the Responder Locus of Drosophila Melanogaster

The Responder (Rsp) locus in Drosophila melanogaster is the target locus of segregation distortion and is known to be comprised of a tandem array of 120-bp repetitive sequences. In this study, we first determined the large scale molecular structure of the Rsp locus, which extends over a region of 600 kb on the standard sensitive (cn bw) chromosome. Within the region, small Rsp repeat arrays are interspersed with non-Rsp sequences and account for 10-20% of the total sequences. We isolated and sequenced 32 Rsp clones from three different chromosomes. The main results are: (1) Rsp repeats isolated from the same chromosome are not more similar than those from different chromosomes. This implies either that there are more homologous exchanges at the Rsp locus than expected or, alternatively, that the second chromosomes of D. melanogaster have diverged from one another more recently at the centromeric heterochromatin than at the nearby euchromatin. (2) The repeats usually have a dimeric structure with an average difference of 16% between the left and right halves. The differences allow us to easily identify the products of unequal exchanges. Despite the large differences between the two halves, exchanges have occurred frequently and the majority of them fall within a 29-bp interval of identity between the two halves. Our data thus support the suggestion that recombination depends on short stretches of complete identity rather than long stretches of general homology. (3) Frequent unequal crossover events obscure the phylogenetic relationships between repeats; therefore, different parts of any single repeat could often have different phylogenetic histories. The high rate of unequal crossing over may also help explain the evolutionary dynamics of the Rsp locus.     

Influence of indole carbinols and growth hormone on the metabolism of 4-androstenedione by rat liver microsomes

The effect of indole-3-carbinol (IC), an anticarcinogen present in cruciferous vegetables, to alter the metabolism of 4-androstenedione (AD) by female rat liver microsomes was investigated and compared to that of its main gastric conversion product, diindolylmethane (DIM) as well as other specific cytochrome P450 inducers. DIM was a more potent inducer of the hydroxylase which converts androsterone to its 6β-hydroxylated derivative 3,6β-dihydroxy-5-androstan-17-one (A) than IC after either oral or intraperitoneal administration and was also a better in vitro inhibitor. Isosafrole (ISF), which like IC and DIM, induces CYP1A2 as well as gestodene, were powerful inhibitors of the in vitro reaction. Naringenin produced only a weak inhibitory effect while 3-methylcholanthrene was inactive. SKF-525A, a prototypic hydroxylase inhibitor, or 17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5-androst-1-ene-3-one which inhibits steroid 5-reductase, also decreased the formation of A from AD by liver microsomes. The infusion of human growth hormone by osmotic minipump, which feminizes hepatic steroid metabolism, increased the ability of male rat liver microsomes to convert AD to A and to respond to induction by IC. The identity of A, the main polar derivative of AD, induced by IC, DIM and ISF, was tentatively assigned by a combination of GC-MS and results from metabolic studies with intermediates in the pathway leading to its formation. It is proposed that the protective role of indole carbinols against mammary carcinoma due to decreased formation of 16-hydroxyestrone from estrone may be further enhanced by the diminished availability of AD for aromatization to estrone.     

FLOW-CYTOMETRIC ANALYSES OF NUCLEAR DNA CONTENT IN FOUR FAMILIES OF NEOTROPICAL BATS

Flow-cytometric analyses of 29 species of microchiropteran bats representing four families and 20 genera revealed that bats possess only 79% (5.43 pg) of the DNA content of a “typical” mammal (e.g., Mus musculus strain C57BL; 7 pg). Chiroptera, the second largest order of mammals, is thus an exception to the prevailing view that mammals possess a minimum nuclear DNA content of 7 pg. Limitations on cell size resulting from a high metabolic rate may have constrained evolution of DNA content and could explain why the extensive heterochromatic additions that are common in some groups of mammals are absent in bats. Chromosomes of bats have been well studied; detailed chromosomal banding data are available for nearly all the species used in this investigation. However, no significant correlations were found between DNA content and karyotypic characteristics such as 2n, fundamental number, and rate or pattern of chromosomal evolution.     

Cytokinins and bud morphology in Pinus radiata

Cytokinins (CKs) play essential roles in the regulation of plant growth and development. In the previous paper (Zhang et al. 2001), we reported the detection and identification of a wide spectrum of CKs, including several novel forms, in the buds of Pinus radiata D. Don. In this paper we examine the relationship between the CKs and buds from juvenile and adult trees of P. radiata. During development the morphology of buds alters significantly, from buds bearing primary needles during their juvenile phase to buds sealed in scales at the adult phase. The morphology of adult buds is a very stable character, as fascicle meristems released from apical dominance, or cultured in vitro, produced only secondary needles. However, exogenous CK causes the adult buds to revert to juvenile bud development in vitro . Analyses of the endogenous CKs revealed that juvenile buds had a relatively higher level of isopentenyladenine and isopentenyladenosine, extremely low levels of phosphorylated CKs and a relatively low level of novel CK glycosides. The adult buds contained lower levels of free base and riboside CKs but very high levels of phosphorylated CKs and novel CK glycosides. Possible roles for CKs in the regulation of bud development are discussed.     

Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen-stigma interactions

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6–8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene ( PCP1 ) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea , Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F₂ segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus .     

The role of temperature in the regulation of the circadian rhythm of CO2 fixation in Bryophyllum fedtschenkoi

Detached leaves of Bryophyllum fedtschenkoi Hamet et Perrier kept in normal air show a single period of net CO₂ fixation on transfer to constant darkness at temperatures in the range 0–25 °C. The duration of this initial fixation period is largely independent of temperature in the range 5–20 °C, but lengthens very markedly at temperatures below 4 °C, and is reduced at temperatures above 25 °C. The onset of net fixation of CO₂ on transfer of leaves to constant darkness is immediate at low temperatures, but is delayed as the temperature is increased. The ambient temperature also determines whether or not a circadian rhythm of CO₂ exchange occurs. The rhythm begins to appear at about 20 °C, is most evident at 30 °C and becomes less distinct at 35 °C. The occurrence of a distinct circadian rhythm in CO₂ output at 30° C in the absence of a detectable rhythm in PEPCase kinase activity shows that the kinase rhythm is not a mandatory requirement for the rhythm of PEPCase activity. However, when it occurs, the kinase rhythm undoubtedly amplifies the PEPCase rhythm.Abbreviation PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea . In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an 'extension' of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of 'self' coating (i.e. that with the same S -haplotype as the stigma) prevent the success of a compatible cross pollination, but a 'cross' coating (i.e. that with a different S -haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen-held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an M_r10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine-rich proteins (PCP-A: P ollen C oat P roteins-class A)—one of which is known to bind to stigmatically-expressed components of the S -locus in Brassica . Introduction of the PCP-A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP-A protein family.     

Stabilized bubbles in the body: pressure-radius relationships and the limits to stabilization

Van Liew, Hugh D., and Soumya Raychaudhuri. Stabilizedbubbles in the body: pressure-radius relationships and the limits tostabilization. J. Appl. Physiol.82(6): 2045-2053, 1997.We previously outlined the fundamentalprinciples that govern behavior of stabilized bubbles, such as themicrobubbles being put forward as ultrasound contrast agents. Ourpresent goals are to develop the idea that there are limits to thestabilization and to provide a conceptual framework for comparison ofbubbles stabilized by different mechanisms. Gases diffuse in or out ofstabilized bubbles in a limited and reversible manner in response tochanges in the environment, but strong growth influences will cause thebubbles to cross a threshold into uncontrolled growth. Also, bubblesstabilized by mechanical structures will be destroyed if outsideinfluences bring them below a critical small size. The in vivo behaviorof different kinds of stabilized bubbles can be compared by using plotsof bubble radius as a function of forces that affect diffusion of gasesin or out of the bubble. The two ends of the plot are the limits forunstabilized growth and destruction; these and the curve's slopepredict the bubble's practical usefulness for ultrasonic imaging orO₂ carriage to tissues.

     

Meiotic chromatin diminution in a vertebrate,the holocephalan fish Hydrolagus collie (Chondrichthyes,Holocephali)

A histochemical, microdensitometric, and electron microscopic study of testes of the ratfish Hydrolagus colliei shows that an instance of the rare phenomenon of germ line chromatin diminution occurs in this vertebrate species. In primary spermatocytes at metaphase I a spherical mass of heterochromatin accumulates at one side of the metaphase plate. At anaphase I the heterochromatic mass is left in the equatorial cytoplasm and is passed into one of the two secondary spermatocytes formed during cytokinesis. As nuclear membranes are being restored, a double membrane envelope is also formed around the heterochromatic mass, which is then termed the ‘chromatin diminution body’ (CDB). At second meiotic division the CDB is included in the cytoplasm of one of the four spermatids and retained there, apparently unchanged, until mid-spermiogenesis. At that time the CDB becomes adherent to the spermatid plasma membrane and is pinched off from the spermatid by a process of apocrine exocytosis, taking a layer of spermatid plasma membrane along with it. Simultaneously this tri-membrane CDB is taken into the adjacent Sertoli cell by endocytosis, thereby acquiring a fourth membrane layer, a part of the Sertoli cell plasma membrane. The CDBs are subsequently phagocytized, possibly first fusing with dense, multilaminate bodies in the Sertoli cell cytoplasm. The CDB chromatin mass is strongly positive with the Feulgen method for DNA and the alkaline fast green method for histones. Microdensitometric analysis shows that the discarded chromatin amounts to about 10% of the diploid nuclear content and that it appears to be part of the normal diploid complement rather than DNA amplified during meiosis.