Rapid isolation of yeast genomic DNA: Bust n' Grab

Background

Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates.

Results

Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC.

Conclusion

An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

     

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.     

Using incidental mark‐encounter data to improve survival estimation

1. Obtaining robust survival estimates is critical, but sample size limitations often result in imprecise estimates or the failure to obtain estimates for population subgroups. Concurrently, data are often recorded on incidental reencounters of marked individuals, but these incidental data are often unused in survival analyses.
2. We evaluated the utility of supplementing a traditional survival dataset with incidental data on marked individuals that were collected ad hoc. We used a continuous time‐to‐event exponential survival model to leverage the matching information contained in both datasets and assessed differences in survival among adult and juvenile and resident and translocated Mojave desert tortoises (Gopherus agassizii).
3. Incorporation of the incidental mark‐encounter data improved precision of all annual survival point estimates, with a 3.4%–37.5% reduction in the spread of the 95% Bayesian credible intervals. We were able to estimate annual survival for three subgroup combinations that were previously inestimable. Point estimates between the radiotelemetry and combined datasets were within |0.029| percentage points of each other, suggesting minimal to no bias induced by the incidental data.
4. Annual survival rates were high (>0.89) for resident adult and juvenile tortoises in both study sites and for translocated adults in the southern site. Annual survival rates for translocated juveniles at both sites and translocated adults in the northern site were between 0.73 and 0.76. At both sites, translocated adults and juveniles had significantly lower survival than resident adults. High mortality in the northern site was driven primarily by a single pulse in mortalities.
5. Using exponential survival models to leverage matching information across traditional survival studies and incidental data on marked individuals may serve as a useful tool to improve the precision and estimability of survival rates. This can improve the efficacy of understanding basic population ecology and population monitoring for imperiled species.

     

Independence of postprandial serum gastrin concentrations on gastric emptying rate in healthy subjects

Changes in serum gastrin concentration after ingestion of meals containing 5 g of guar gum or 5 g wheat flour were studied in 15 healthy subjects for up to 120 minutes postprandially. The results showed that the postprandial gastrin response was independent of changes in gastric emptying rate. A two-fold increase in serum gastrin concentration prevailed for 60 to 90 minutes postprandially. In addition, was serum gastrin concentration was independent of the amount of food in the stomach.     

Identified taste bud cell proliferation in the perihatching chick [published erratum appears in Chem Senses 1998 Aug;23(4):459]

Developing taste buds in the anterior mandibular floor of perihatching chicks were studied by high voltage electron microscopic autoradiography in order to identify proliferating gemmal cell types. Montaged profiles of 29 taste buds in five cases euthanized between embryonic day 21 and posthatching day 2 were analyzed after a single [3H]thymidine injection administered on embryonic day 16, 17 or 18. Results showed that dark cells comprised 55% of identified (n = 900 cells) and 62% of labeled (n = 568 cells) gemmal cells as compared with light, intermediate, basal or perigemmal bud cells. Dark cells had both a greater (P < 0.05) number of labeled cells and a greater amount of label (grains/nucleus) than the other four bud cell types, irrespective of injection day. The nuclear area (micron 2) of dark cells was not significantly larger (P > 0.05) than that of the other gemmal cell types and therefore cannot account for the greater amount for label in the dark cells. Interestingly, only dark cells showed a positive correlation (P < 0.003) between amount of label and nuclear area. Results suggest that, during the perihatching period of robust cell proliferation, dividing dark cells may give rise primarily, but not exclusively, to dark cell progeny.