Science & Mathematics

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine. Either hepatic DNA was prelabeled with [³H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase of single-stranded DNA up to 1.0 kilobase in length. The data suggest that structural lesions in heterochromatin, which may be a consequence of incomplete repair, are preferentially degraded by endogenous nuclease(s)

Different levels of damage and repair to eu- and heterochromatic DNA from the livers of rats receiving a dose of 10 mg/kg N-nitrosodimethylamine (NDMA) were apparent. Preincorporated ³H~thymidine was lost rapidly from euchromatic DNA but persisted in the heterochromatic fraction. Persistent damage, determined as single-stranded regions binding 10 benzoylated DEAE-cellulose (BD-cellulose), was evident in heterochromatic DNA for up to three months. By subjecting rats treated with NDMA to partial hepatectomy, generation of single-stranded regions in the newly synthesized heterochromatic DNA could be demonstrated. Such structural defects were evident when hepatectomy was performed two months after administration of the carcinogen. These findings indicate that structural damage to non-transcribed DNA is one of the most persistent molecular lesions following exposure to nitrosamines.

We have investigated structural change in rat liver DNA produced by different isolation procedures and specifically compared the integrity of DNA derived by phenol extraction from isolated and purified nuclei with preparations extracted immediately from a crude liver homogenate containing intact nuclei. As indicated by stepwise elution from benzoylated DEAE-cellulose, most structural change in DNA was evident following nuclei isolation. Damage principally involved generation of single-stranded regions in otherwise double-stranded DNA fragments; totally single-stranded DNA was not detected by hydroxylapatite chromatography. Caffeine gradient elution suggested formation of single-stranded regions extending for up to several kilobases. In neutral sucrose gradients, differences in sedimentation rates of respective DNA samples consequent upon S1 nuclease digestion could be detected after isolation of nuclei, though not in other circumstances. The observed single-strand-specific nuclease digestion of DNA could apparently be reduced if steps were taken to reduce autodigestion during nuclei isolation by reduction of temperature and covalent cation concentration. The results are discussed in terms of the use of exogenous and endogenous nucleases in chromatin fractionation studies involving isolated nuclei and possible artifactual findings that may be generated by single-strand-specific autodigestion.

The proportion of sheared rat liver DNA recovered from benzoylated DEAE-cellulose in the final stage following stepwise elution with NaCl and caffeine solutions was dependent upon the DNA isolation procedure. An increase in the proportion of DNA containing single stranded regions, consequent upon delay or addition of Mg2+ prior to phenol extraction, suggested nuclease mediated degradation. Administration of methyl methanesulphonate to rats resulted in a consistent proportional increase in the caffeine-eluted fraction. The results of caffeine gradient elution of control and alkylated DNA from benzoylated DEAE-cellulose were consistent with repair-associated single stranded regions being substrates for endogenous single strand-specific exonucleases.

The orchid genus Rhizanthella is endemic to Australia and comprises two species, R. gardneri, which is known from a number of sites in S. W. Western Australia and R. slateri, which is now known from six confirmed sites in eastern Australia. One of the most remarkable characteristics of the genus is that almost the entire life cycle is spent underground.

750 reptile specimens comprising 38 species, representing 30 genera and 7 families were examined from the Wagga Wagga area. A further 9 species might be expected to occur in the area or nearby regions. Two species not previously recorded from the area, Gehyra variegata and Lerista muelleri are south-east range extensions for these species. Significant habitat alteration in the area has probably resulted in reduction in reptile numbers and the species densities. Fractionation of habitat has resulted in the isolation of many populations.

Observations on the small, specialised, fossorial and legless Punctuate Work-skink (Anomalopus (Vermiseps) swansoni) both in captivity and of museum specimens show: 1. A patchy distribution but with a range extension to the north-west. 2. Exclusive or near exclusive termite feeding. 3. Production of two or three young in late summer. 4. Possible thermoregulatory behaviour. 5. A longevity of at least ten years. 6. An indeterminate conservation status.