Progeny testing for recessive genes: procedures and interpretations

Summary Methods for calculating the probability of detecting a carrier of a recessive gene by utilizing matings among related individuals are presented for single and litter bearing species. The confidence level for detection of heterozygosity depends upon: (1) the genetic relationship between mates, (2) the number of mates per male and the number of offspring per mate, (3) whether an estimate of recessive gene frequency before selection is available and (4) the magnitude of that frequency. Methods of computing probability of heterozygosity vs homozygosity utilizing Bayes theorem also are presented. In the conventional progeny test method, a sire initially is assumed heterozygous before calculations are made, but no prior information concerning his probable genotype is utilized. In the method using Bayes theorem, prior sources of information from relatives or from estimates of population allele frequency are utilized. This method gives the exact probability that a sire is not a carrier, given prior information and that he produces all normal offspring. These methods could be used in any sexually reproducing species to identify not only detrimental genes but beneficial genes as well.     

Use of a Novel Type of Rotating Disc Electrode and a Flow Cell with Laminar Flow Pattern for the Electrochemical Detection of Biogenic Monoamines and Their Metabolites After Sephadex Gel Chromatographic Purification and High Performance Liquid Chromatographic Isolation from Rat Brain

Abstract: A novel type of rotating disc electrode and a flow cell with laminar flow pattern were developed and applied to the electrochemical detection of dopamine, 3,4-dihy-droxyphenylacetic acid, homovanillic acid, 3-methoxytyra-mine (3-MT), noradrenaline, 3-methoxy-4-hydroxyphenyl-ethyleneglycol (MOPEG), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid after HPLC of these compounds. The active surface of the rotating disc working electrode was made from solid paraffin (40%; wt/wt) and graphite powder (60%; wt/wt). The sensitivity of the detector was proportional to the square root of the angular velocity and was practically independent of the flow rate of the mobile phase. The surface of the working electrode was very large (radius = 12 mm), and so the percentage of oxidation was 24–67%; (flow rate = 1.0 ml/min), depending on the compound. Electrical noise between 20 and 40 pA and background current of 20–60 nA were observed. In practice, the sensitivity for the detection of the compounds examined here was 8–16 nA/ng, and so a detection limit of 5 pg/injection could be achieved, when the detector was combined with reversed-phase HPLC. Supernatants obtained from the extracts of the tissue samples (nine brain parts of rat brain were studied) were purified by using Sephadex G-10 gel chromatography. Before this procedure, the proteins of the tissue extracts were precipitated by 0.2 M HC1O₄, and the excess of HC1O₄ was precipitated by KOH/HCOOH buffer. Simultaneously, the pH of the extracts was set to 2.4 by the above buffer. Adjustment of the pH was necessary so that elution of 5-HT from the Sephadex G-10 columns in the same fraction with 3-MT was avoided. If these compounds were in the same solution, their peaks would overlap on HPLC. MOPEG sulfate was purified by diethylaminoethyl-Sephadex A-25 (anion exchange resin) from the first fraction collected from the Sephadex G-10 columns. The contents of the compounds under investigation in nine brain parts agreed with those found by other investigators.     

Diagnosis of early pregnancy by ultrasound in Macaca fascicularis

Real-time ultrasonography was used to detect early pregnancy in 32 longtailed macaques (Macaca fascicularis). In 92% of the successful conceptions, a correct diagnosis was made. The earliest sign of pregnancy was an intrauterine ringlike structure (11 days). A "line swelling" (14 days) preceded definite fetal echoes (21 days), and fetal heart motion (30 days) proved fetal viability. Ultrasound is a rapid, noninvasive, and relatively cost-effective method of diagnosing and monitoring early pregnancy in M. fascicularis.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Analysis of DNA-protein complexes induced by chemical carcinogens.

DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.     

Non-radioactive detection of single-copy DNA-DNA hybrids

A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.     

Detecting estrus in synchronized heifers-using tailpaint and an aerosol raddle

A synchronization treatment was initiated when each of 1227 heifers (four trials) was tailpainted. The tailpaint was sprayed with an aerosol raddle at the end of the treatment period. The heifers were in herds of 20 to 279 animals. Each herd was observed for estrus at selected post treatment intervals. A heifer was considered to be (or to have been) in estrus when the raddle was rubbed off. In three of the trials, animals which had the raddle removed were inseminated at 48h following the end of the synchronization treatment. The tailpaint of an inseminated animal was scored from 0 (less than 10% of the paint remained) to 5 (more than 90% of the paint remained) and was then reraddled with a second color. The detection-insemination sequence was always repeated at 72 and 96h, and sometimes at 120h. Animals which had been previously inseminated, but then had paint scores reduced by at least 2 units were reinseminated 24h later. Over the four trials, 94.5% of the heifers were detected in estrus through the use of the tailpaint and raddle system. The remaining 67 animals included only 10 (0.8%) which had ovulated without being detected in estrus. The reinsemination rate on consecutive days was 11.3% and was highest among animals that had a tailpaint score of 4 or 5 at 48h. The proportion of animals detected in estrus at selected posttreatment intervals varied with the different synchronization treatments used within one herd, or with the same treatment used in different herds. The combination of tailpaint, raddling, tailpaint scoring and reraddling is a simple sequence which can be effectively used to detect estrus among heifers synchronized in research or commercial herds.     

Anti-bromodeoxyuridine monoclonal antibody: an alternative tool for the identification of replicated DNA at the electron microscope level

A new method for identifying the replicated DNA at the electron microscope level is described. Cells were first exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with 5-fluorodeoxyuridine (FUdR) and BUdR incorporated into DNA was then detected on Lowicryl-embedded sections by immunogold technique using a monoclonal anti-BUdR antibody. After using this method, chromatin and chromosomes are strongly labelled.     

Fast HPLC Estimation of γ-Aminobutyric Acid in Microdialysis Perfusates: Effect of Nipecotic and 3-Mercaptopropionic Acids

A method for rapid, automated (less than 5 min), and sensitive (detection limit 50 fmol/10 microliter) determination of gamma-aminobutyric acid (GABA) is described. The method is based on precolumn derivatization with o-phthaldialdehyde/t-butylthiol reagent and separation by reverse-phase HPLC with electrochemical detection under isocratic conditions. A 100 X 4 mm Nucleosil 3 C18 column was used; the mobile phase consisted of 0.15 M sodium acetate, 1 mM EDTA (pH 5.4), and 50% acetonitrile; the flow rate was 0.8 ml/min. The potential of the glassy carbon working electrode was +0.75 V. The method allows for the monitoring of GABA levels in the extracellular fluid sampled by microdialysis as documented in the present study when 0.5 mM nipecotic acid is infused via the probe, or 3-mercaptopropionic acid is injected at a dose of 100 mg/kg i.p. There was a 15-fold increase of extracellular GABA after nipecotic acid, whereas in the second case the inhibition of GABA synthesis was followed by a 74% decrease of GABA as compared to basal levels.