Endo-exonuclease of Aspergillus nidulans

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.     

Differential methylation of the hypervariable locus DXS255 on active and inactive X chromosomes correlates with the expression of a human X-linked gene

Consistent differences in methylation of particular cytosine residues in the DNA of active and inactive X chromosomes can be used for rapid, direct analysis of X-inactivation patterns in different female tissues. We have studied methylation of the highly polymorphic DXS255 locus in tissues from patients with deficiency of the E1 alpha subunit of the pyruvate dehydrogenase complex in whom the results can be correlated directly with total enzyme activity, levels of immunoreactive protein, and patterns of cell mosaicism. The results confirm that methylation of the DXS255 locus correlates with X-chromosome expression. In patients and normal controls, the pattern of X inactivation varied widely from tissue to tissue and often deviated markedly from a 50:50 proportion. These deviations are likely to reflect small numbers of tissue-specific stem cells at the time of random X inactivation and cannot be taken alone as evidence for selection or "nonrandom" inactivation.     

Dynamic changes in optic fiber terminal arbors lead to retinotopic map formation: an in vivo confocal microscopic study

Dynamic remodeling of retinal ganglion cell terminal arbors has been proposed to contribute to formation of the topographically ordered retinotectal projection. To test this directly, the growth of individual terminal arbors was observed in live X. laevis tadpoles using a confocal microscope to visualize their complex three-dimensional structure. During initial development, nasal and temporal retinal arbors covered overlapping tectal areas. Despite subsequent remodeling, the dimensions and positions of the temporal arbors remained relatively stable. In contrast, the nasal arbors grew caudally, as they extended caudal branches and retracted rostral branches. These results suggest that differences in the remodeling of the nasal and temporal arbors lead to the emergence of retino-topography along the rostrocaudal axis of the tectum. All the terminal arbors were dynamic, including those with stable dimensions, suggesting that continual remodeling of arbors may be a universal feature of neuronal projections.     

Uptake and degradation of hyaluronan in lymphatic tissue.

Afferent lymph vessels entering popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan of high Mr (4.3 x 10(6)-5.5 x 10(6)) and low Mr (1.5 x 10(5)). Analysis of efferent lymph and of residues in the nodes showed that hyaluronan presented by this route is taken up and degraded by lymphatic tissue. Labelled residues isolated in node extracts by gel chromatography and h.p.l.c. included N-acetylglucosamine, acetate, water and a fraction provisionally identified as N-acetylglucosamine 6-phosphate. Between 48 and 75% of the infused material was unrecovered, and had been presumably eliminated through the bloodstream as diffusible residues. Rates of degradation reached as high as 43 micrograms/h in a node of 2 g wt. infused with 56 micrograms/h. Some HA passed into efferent lymph and some was detected in the nodes, but fractions of Mr greater than 1 x 10(6) were not found in either. It is concluded that the amounts and Mr values of hyaluronan released from the tissues into peripheral lymph can be significantly underestimated by analysis of efferent lymph, i.e. lymph that has passed through lymph nodes. A substantial role in the normal metabolic turnover of at least one major constituent of intercellular matrix and connective tissue may now be added to the established functions of the lymphatic system.     

Effect of straw on the behaviour of growing pigs

The effects of straw on the behaviour of young growing pigs were studied in two experiments. In Experiment 1, groups of three pigs, aged about 7 weeks, were housed in raised decks with or without straw bedding. Time-lapse video recording showed no major differences between treatments in the amount or daily pattern of overall activity and feeding. Detailed observations showed that bedding reduced the incidence of rooting and chewing on pen-mates, but had little effect on other social activities such as mounting and aggressive biting. Experiment 2 compared groups of eight growing pigs aged about 10 weeks, housed in floor pens with or without a small amount of straw provided daily in a rack. Pigs with straw concentrated more of their daily activity into the period when straw was fresh, but the total amount of overall activity and time spent feeding were not affected. Rooting and chewing of pen-mates were the only social activities reduced by the provision of fresh straw. In these studies, where straw was not required to compensate for deficiencies such as low temperatures or hunger, the one major function of straw was to provide a stimulus and outlet for rooting and chewing, with a resulting reduction in such activities directed at pen-mates.     

Parthenogenetic stem cells in postnatal mouse chimeras.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)