Colony hybridization method for screening in situ of eukaryotic amplified genes

Summary A new method for autoradiographic screening of amplified genes in cellular clones is described. The main feature of the device is to keep viable cells from each clone, which can subsequently be regrown. The availability of this biochemical screening method allows screening for recombinants harboring unselectable markers as well.     

Localization of amplified DNA in nuclei of the orchid Cymbidium by in situ hybridization

Summary In situ hybridization of³H-polyuridylic acid of low specific activity to somatic nuclei of Cymbidium protocorms is suggested to indicate the location of the highly amplified AT-rich DNA fraction.     

A method for the destructionin situ of gonads ofDrosophila larvae

Zusammenfassung An männlichen und weiblichen Drosophilalarven im dritten Larvenstadium lassen sich die Gonaden durch Hindurchleiten eines hochfrequenten Wechselstroms (Frequenz 20 000 Hz, Stromstärke empirisch) ohne Schädigung anderer Organein situ zerstören. Die Methode ist bei Männchen erfolgreicher als bei Weibchen. Die Versuchsanordnung und die Herstellung einer geeigneten Mikroelektrode werden beschrieben.     

An improved electrophoretic method for a screening program for haemoglobinopathies

Summary A method for a screening program for haemoglobinopathies in a starch agar gel mixed with saponin is presented. Normal and abnormal blood containing haemoglobins S, C, I, M Boston, D Punjab, beta thalassaemia major and beta thalassaemia minor, were applied, in a tray with the capacity for 100 samples. The electrophoresis was performed in 45 min using 300 V. This method offers special advantages for the examination of a large number of samples, using a small amount of whole blood and without the previous preparation of haemoglobin solution.Acknowledgments. We would like to express our gratitude to Dr C. Daghlian and Dr Lewis J. Greene for their valuable suggestions. We also thank the Conselho Nacional do Desenvolvimento Científico e Tecnológico, CNPq, for financial support.     

In situ hybridization analysis of globin mRNAs in the primitive erythroid cells of the chick embryo

The possibility that the minor embryonic chick hemoglobins might be present in a particular subgroup of primitive erythroid cells has been investigated by in situ hybridization. Probe to detect the mRNA for the ^A globin chain of the minor embryonic hemoglobin was used, and the results of the hybridization were compared with those obtained using as probes the cDNAs for total globin mRNAs. All erythroid cells circulating in a 4-day-old chick embryo gave positive signals with both probes at an approximately constant ratio. This shows that all cells contain a similar assortment of hemoglobin types, excluding the possibility that a subgroup might contain the minor primitive hemoglobins exclusively. However, the cells are not homogeneous, since about 10% of them show a distinctly higher concentration of mRNA of all globin types.     

In situ hybridization of rDNA on chromosomes of the honeybee,Apis mellifera L.

DNA probes containing the repeated rDNA region ofDrosophila melanogaster (coding for e.g. 28S and 18S rRNA) hybridized in situ to distinct regions of two heterologous mitotic chromosomes of the honeybee, identifying the nucleolus organizing regions (NORs). The method allows a rapid establishment of a physical map ofApis mellifera using other DNA probes ofDrosophila. This is the first report on well-defined chromosomal markers in the honeybee.     

A turbidimetric method for the screening of amylase-producing mutants of Aspergillus niger

Summary The method is based on the assumption that extracellular amylase, which is produced by strains ofAspergillus niger in liquid culture, hydrolyses the starch in the media and brings about a corresponding decrease in the turbidity of the media. Mutant strains which produced different quantities of amylase exhibited different degrees of decrease in turbidity of the media. The results showed that a greater degree of decrease in turbidity was observed for a higher quantity of amylase produced.     

A Y-translocation method for localizing enzyme genes onDrosophila polytene chromosomes

Summary X-Ray induced translocations between autosomes and the Y-chromosome giving balanced and aneuploid (partially trisomic) male offspring proved useful for a rather precise localization of enzyme loci in the subsections of the polytene chromosomes ofDrosophila subobscura.Acknowledgments. The work was supported by the Deutsche Forschungsgemeinschaft (Projekt Sp 146-6/1). We are very much obliged to Miss I. Kaipf and to Mrs. K. Stögerer for their excellent technical help.     

The emerging role for sphingolipids in the eukaryotic heat shock response

Eukaryotic cells have a highly conserved response to an increase in temperature, termed the heat shock response. Recent research has revealed multiple roles for various sphingolipids in the heat shock responses of both yeast and mammalian cells. Heat stressed or shocked yeast and mammalian cells have an acute activation of serine palmitoyltransferase, resulting in the de novo biosynthesis of sphingolipids. Also, both mammalian and yeast cells were shown to increase ceramide levels upon heat stress or shock. In yeast cells, several functions have emerged for the de novo produced sphingoid bases in terms of the heat stress response. These functions include a role in accumulation of trehalose, a role in the heat-induced transient G0/G1 cell cycle arrest and phytosphingosine activation of a ubiquitin protein degradation pathway. However, in mammalian systems, ceramides have been demonstrated as bioactive lipids. Ceramides produced in response to heat shock were demonstrated to induce the production of c-jun, leading to apoptosis, and to be upstream of dephosphorylation of serine-rich proteins. Increasingly, sphingolipids are emerging as bioactive signaling molecules involved in numerous aspects of the eukaryotic heat shock response.     

Evolution of intrinsic disorder in eukaryotic proteins

Conformational flexibility conferred though regions of intrinsic structural disorder allows proteins to behave as dynamic molecules. While it is well-known that intrinsically disordered regions can undergo disorder-to-order transitions in real-time as part of their function, we also are beginning to learn more about the dynamics of disorder-to-order transitions along evolutionary time-scales. Intrinsically disordered regions endow proteins with functional promiscuity, which is further enhanced by the ability of some of these regions to undergo real-time disorder-to-order transitions. Disorder content affects gene retention after whole genome duplication, but it is not necessarily conserved. Altered patterns of disorder resulting from evolutionary disorder-to-order transitions indicate that disorder evolves to modify function through refining stability, regulation, and interactions. Here, we review the evolution of intrinsically disordered regions in eukaryotic proteins. We discuss the interplay between secondary structure and disorder on evolutionary time-scales, the importance of disorder for eukaryotic proteome expansion and functional divergence, and the evolutionary dynamics of disorder.     

Marked by association: techniques for proximity-dependent labeling of proteins in eukaryotic cells

Various methods have been established for the purpose of identifying and characterizing protein–protein interactions (PPIs). This diverse toolbox provides researchers with options to overcome challenges specific to the nature of the proteins under investigation. Among these techniques is a category based on proximity-dependent labeling of proteins in living cells. These can be further partitioned into either hypothesis-based or unbiased screening methods, each with its own advantages and limitations. Approaches in which proteins of interest are fused to either modifying enzymes or receptor sequences allow for hypothesis-based testing of protein proximity. Protein crosslinking and BioID (proximity-dependent biotin identification) permit unbiased screening of protein proximity for a protein of interest. Here, we evaluate these approaches and their applications in living eukaryotic cells.     

Progress in screening for inborn errors of metabolism

Summary The inborn errors of metabolism are a series of individually rare biochemical anomalies some of which cause serious clinical manifestations. They are of great interest to biochemists and geneticists, as well as to paediatricians and internist for whom they often present special diagnostic and therapeutic problems. The study of the inborn errors of metabolism also has implications in the fields of epidemiology and social medicine. The number of known inborn errors of metabolism has increased rapidly in recent years, and others, as yet unidentified, presumably await recognition. Only a few of these conditions can be treated now, but the realisation that early diagnosis is essential in order to achieve good results has stimulated interest in the possibility of examining either whole populations or selected predisposed groups of individuals for biochemical differences which characterise particular inherited metabolic diseases.This article reviews some recent developments with particular reference to the indications for such screening programmes and progress in the identification of previously unknown inborn errors of metabolism in otherwise homogeneous population groups. — The inborn errors of metabolism are due to single gene mutations. — Recognition of the asymptomatic individuals who are heterozygous for the abnormal gene causing the disease may be important clinically and the identification of these individuals has to be considered as one aspect of metabolic screening for the inborn errors of metabolism.     

Biological aspects of cytosine methylation in eukaryotic cells

The existence in eukaryotes of a fifth base, 5-methylcytosine, and of tissue-specific methylation patterns have been known for many years, but except for a general association with inactive genes and chromatin the exact function of this DNA modification has remained elusive. The different hypotheses regarding the role of DNA methylation in regulation of gene expression, chromatin structure, development, and diseases, including cancer are summarized, and the experimental evidence for them is discussed. Structural and functional properties of the eukaryotic DNA cytosine methyltransferase are also reviewed.