A simple banding technique for identification of human metaphase chromosomes

Summary A simple technique of Giemsa staining is described giving characteristic banding patterns of human metaphase chromosomes; these patterns can be used to identify all 24 chromosome types.

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Zusammenfassung Wir beschreiben eine einfache Methode, die mit geringem Zeitaufwand charakteristische Bandenmuster in menschlichen Metaphase-Chromosomen ergibt. Die Muster ermöglichen die Identifizierung aller 24 Chromosomentypen.

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Director: Prof. Dr. W. Fuhrmann

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Supported by the Deutsche Forschungsgemeinschaft and the Sonderforschungsbereich 35.     

A rapid assay of urinary estrogens of the Japanese monkey for routine use

A simplification of Brown’s fluorometry for monkey urinary estrogens has been attained. The method requires only a one-step extraction of estrogens instead of the complicated three-step extractions in the original Brown’s method. The procedure made it possible to shorten the time from the six to seven hours required in the original method to three hours. The present method is satisfactory in precision, sensitivity, accuracy, and specificity. Although the values of urinary estrogens obtained by the simplified method are lower than those obtained by the original method, the use of the present method is validated, since the correlation coefficient between the two assay procedures is excellent (γ=0.90) and also the application of the present method to the measurement of daily changes in urinary estrogens throughout the menstrual cycle has been successful. The simplified assay is expected to be useful in the routine assessment of urinary estrogens in Japanese monkeys. This work was supported by a grant from the Ford Foundation (No. 670-0558).     

A new R-banding technique in clinical cytogenetics

Summary Currently, standard methods of chromosome banding detect an abnormality in some 10%–15% of all patients referred for a cytogenetic study. Higher resolution by the study of the less contracted chromosomes with or without cell synchronization techniques may yield higher frequencies in the future. On the other hand, use of more complicated methodology adds to the time and expense needed for the study. The method of 5 bromodeoxyuridine pretreatment of cells in culture is commonly used to study the replication behavior of X chromosomes, and is known to demonstrate R bands along the other chromosomes. We have modified this technique with the addition of a cell synchronization step and evaluated several variables that are of importance in a clinical service laboratory setting. The method offers a simple way to obtain quality R-banded karyotypes. The advantages and limitations, based on our study of 120 consecutive cultures, are described.This work was presented in part at the 1979 Birth Defects Conference, Chicago     

A versatile microrespirometer for routine use

A new design of microrespirometer suitable for routine laboratory work has been described.     

A new technique for orcein banding with acid treatment

A rapid method for banding plant chromosomes using hydrochloric acid with orcein is suggested. The technique has been successful in both dioecious and monoecious families with short chromosomes. It involves pretreatment with an oxyquinoline-paradichlorobenzene mixture, fixation in acetic ethanol and treatment with hydrochloric acid at 60 C followed by staining with orcein. Stained chromosome bands and interbands are reproducible and species specific.     

Interphase cytogenetics and comparative genomic hybridization of human epithelial cancers and precursor lesions

 The accuracy of cytogenetic analyses of human solid cancers has improved enormously over the past decade by the introduction and refinement of DNA in situ hybridization (ISH) techniques. This methodology can be applied to cells in the interphase state, thereby making it an excellent tool for the delineation of chromosomal aberrations in solid tumors. The use of non-isotopic ISH to intact and disaggregated cancer specimens will be discussed, as well as comparative genomic hybridization (CGH) with tumor-derived DNAs. In this review we will focus on hybridocytochemical interphase approaches for the detection of chromosomal changes in frequently occurring human epithelial malignancies, e.g., breast, lung, and prostate carcinomas. We will further discuss the use of ISH procedures for the genetic analysis of precursor conditions leading to invasive carcinomas. Knowledge concerning these precancerous conditions is increasing, and its importance in cancer prevention has been recognized. Interphase cytogenetics by ISH, as well as CGH, with DNAs derived from microdissected, precancerous, dysplastic tissue areas will increase our understanding of these lesions, both at the investigative and diagnostic levels. Accepted: 27 June 1997     

A new rapid radiological procedure for routine teratological use in bone ossification assessment: a supplement for staining methods

BACKGROUND: Presently, bone ossification is assessed by the study of single-stained fetal bones (alizarin red-S) or double-stained bones and cartilaginous structures (alcian blue followed by alizarin red-S). Both methods, especially double-staining, are labor-intensive, time-consuming, and provide qualitative information regarding skeleton ossification. Quantitative evaluation of ossification is more difficult and is usually based on determination of calcium and other minerals in the bone by means of atomic absorption spectrometry. Here we introduce a simple new method that allows quantitative determination of skeleton ossification before routine staining examination. METHODS: Fetuses delivered by laparotomy on the 16th and 21st day of gestation as well as 1-day-old rat pups were examined. The fetuses and pups were prenatally subcutaneously exposed to sodium valproate or to physiological saline. Lateral, prone, and supine digital radiograms of each fetus were taken using the Digora-Soredex digital radiography system and the Planmeca Intra intraoral X-ray machine. According to the best visualization, the data concerning vertebra were analyzed. All the fetuses were then routinely double-stained using alcian blue and alizarin red-S. RESULTS: Malformations of axial skeleton (rib, sternum, and thoracic and sacral vertebra) were found in valproate-treated groups. Unlike cartilage malformations, the bone changes were detected in similar frequency in radiological and staining methods. Differences in densities according to the degree of ossification in the vertebral arches and bodies at different levels of the vertebral column, between drug-treated and negative control groups were noted. CONCLUSIONS: The preliminary results suggest that digital radiography examination is a useful method in determining delaying of skeleton ossification not detectable by other methods. It balances qualitative and quantitative aspects of the presently used methods and is also simple, objective, fast, and relatively inexpensive.     

A rapid radioassay technique for cellular suspensions

A simplified technique for studying in vivo the uptake of labeled precursors into proteins and nucleic acids of microorganisms is presented. Applications to studies in protozoan and bacterial systems are represented. The details of the technique are discussed along with drawbacks and advantages.     

A simple and rapid technique for assaying toxicants in aphids

A simple bioassay technique has been developed in order to study the effect of toxic compounds on aphids. The technique involves dipping of leaves of a particular aphid host in a solution of the desired toxic compound contained in a vacuum flask. The flask is then connected to a vacuum source in order to remove the air from the intercellular spaces of the palisade and the spongy parenchymas of the leaves. Breakage of the vacuum and return to atmospheric pressure forces the test solution into the now empty intercellular spaces which are thus filled with the solution. The treated leaves exhibited a typical water-soaked appearance. The infiltrated leaves were then offered to the aphids. The above treatment did not affect either the structure of the leaves or the development of the aphid colony on them. Aphids grown on leaves infiltrated with water lived longer in comparison with controls. In addition, insects grown on leaves infiltrated with 27.5, 13.7 and 6.9 p.p.m. of the systemic insecticide heptenophos died after 1.5, 3.0 and 5.0 h, respectively. The bioassay technique described in this report has certain advantages: it is simple, fast and inexpensive and is especially suitable for short-term studies involving the effects of such chemicals as toxins, insecticides and other toxicants on aphids. It may also be used to study the effects of growth factors, hormones and special nutrients on aphid growth and possibly on other sucking insects.     

A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells

We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (10⁵) for any target sequence for use in human cells. In this system, target sequences are fused to the 5′ end of the lacZ reporter gene and expressed in yeast. Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype. We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors. GSCs specific for c-myc were shown to regulate expression of both a c-myc–lacZ fusion gene and the endogenous c-myc gene in human cells.