Dale M. Steffensen
University of Illinois at Urbana–Champaign
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Featured researches published by Dale M. Steffensen.
Journal of Molecular Biology | 1977
Paul Szabo; Robert T. Elder; Dale M. Steffensen; Olke C. Uhlenbeck
Abstract In situ hybridization of 125 I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ . This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.
Chromosoma | 1974
D. E. Wimber; Patricia A. Duffey; Dale M. Steffensen; W. Prensky
Abstract5S RNA was extracted from Zea mays tissue and iodinated in vitro with 125I to a high specific activity. Acrylamide gel electrophoresis of the 125I-5S RNA, 11/2 weeks after iodination demonstrated that most of the 5S RNA molecules were degraded to half-size or smaller. In situ hybridization with this iodinated RNA to pachytene microsporocyte chromosomes showed that the 5S RNA cistrons are located near the end of the long arm of chromosome 2. No obvious association of the 5S locus with the nucleolus was seen during pachytene or later stages.
Human Genetics | 1977
Dale M. Steffensen; Ernest H. Y. Chu; David P. Speert; Patrick M. Wall; Karen Meilinger; Robert P. Kelch
SummaryIn a newborn boy with multiple malformations, a tandem duplication was detected at the distal end of the long arm of one human chromosome 1. The Giemsa bands, 1q31 to 1q43–44, were repeated serially. Since 5S rRNA genes are located at 1q42–43, in situ hybridization of 125I 5S rRNA with fixed chromosome preparations was used to confirm the chromosomal duplication. The infant exhibited numerous developmental and clinical abnormalities as might be expected with an abnormality of chromosome structure relating to a ribosome component.
Analytical Biochemistry | 1968
Robert H. Gray; Dale M. Steffensen
Abstract A new method is described for slicing polyacrylamide gels and couting radioactivity in macromolecules separated by disc electrophoresis. Polyacrylamide gels are dehydrated and sectioned uniformly with a rotary microtome into slice thicknesses of 250, 125, 62.5, or less than 50 μ. The gel slices are solubilized in hydrogen peroxide and counted in Bray solution. The counting efficiency for 14C and 35S is about 90% and about 33% for 3H. The method is also applicable for analyses of double-labeled compounds in polyacrylamide gels.
Chromosoma | 1981
Dale M. Steffensen; R. Appels; W. J. Peacock
In situ hybridization using 3H-RNA probes has been used to localize the sequences found in two satellites of density 1.705 g/cc and 1.672 g/ cc to specific sites within the chromosomal complement. A detailed analysis of the sites on the X chromosome was carried out using the scute series of inversions to relate the heterochromatic breakpoint relative to the location of the sequence on this chromosome. It has also been possible to establish the order of arrangement of 1.705 and 1.672 DNA at the heterochromaticeuchromatic junction on chromosome 3(R). A mitotic map is provided. The Tm of hybrids formed in situ showed that the hybrids were representative of the sequences being analyzed. The two satellites also were traced through a number of purification procedures to show that a covalent linkage may be likely between the 1.705 g/cc and 1.672 g/cc satellite as predicted from in situ hybridization analyses.
Chromosoma | 1980
Babette D. Coté; Olke C. Uhlenbeck; Dale M. Steffensen
The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitate the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.
International Review of Cytology-a Survey of Cell Biology | 1987
Ronald J. Hill; Margaret R. Mott; Dale M. Steffensen
Publisher Summary The polytene chromosomes prepared by the acid-squashing technique preserve morphology for study at the light microscope level and have played a major role in the development of the concepts of genetics over the past half century. Acid squashing is the method of choice for preparation of chromosomes for in situ hybridization to localize DNA sequences; in this approach strand separation is mandatory to allow hybridization. However, when in situ hybridizing to nascent RNA the possibility of introduction of some DNA hybridizability should be kept in mind during acid squashing without a deliberate subsequent “denaturation” step. This phenomenon may be locus-specific depending on the local “state” of the chromatin. When DNA sequences are to be isolated by microcloning, it is important to minimize exposure to acid fixatives during chromosome preparation if depurination is not to become a serious complication. The chapter explores that the chromosomes prepared without exposure to acid fixatives appear to simultaneously present optimal resolution of banding patterns and preservation of ultrastructural details.
Pollen#R##N#Development and Physiology | 1971
Dale M. Steffensen
Publisher Summary This chapter discusses the comparative study of ribosome synthesis, dealing with the other two components, the ribosomal proteins and 5S RNA. Microspores in detached buds were labeled in vitro with 14CO2 at the peak of ribosome synthesis. By contrast, if pollen tubes were labeled with 35S and the ribosomes were extracted, the resulting ribosomal proteins are not radioactive at all nor are the histones. The pollen tube offers a unique system to study different RNA molecules because the noise from ribosome synthesis is shut off. The most interesting finding about tRNA concerns its methylation in the pollen tube when 3H-methyl methionine is used. A finding that was unexpected was that pectin synthesized in pollen tubes can be isolated on a methylated albumin on kieselguhr column just before the tRNA begins to elute with salt.
Biochemical Genetics | 1987
Laura M. M. Ottoboni; Dale M. Steffensen
The band patterns of zein polypeptides were determined for many commercial inbred corn lines and maize stocks using isofocusing in agarose gels and sodium dodecyl sulfate (SDS)-urea gels. Each inbred line or homozygous maize strain genotype has a distinct zein profile which has been catalogued according to the distance of charge migration and molecular weight (kilodaltons). Several zein polypeptides were mapped to chromosomes 4 and 10 with the use of reciprocal translocations. The mapping of at least two polypeptides on distal 4L and 10L had not been previously reported. The general methods used in the present research will permit the mapping of all the zein polypeptides to chromosomal sites.
Mutation Research | 1968
Dale M. Steffensen
Abstract The frequency of losses of chromosome markers, attributable to calcium-45 or to strontium-89, was found to be nil in the endosperm of maize that develop from pollen labeled at the isotope levels employed and in the population sizes examined. For comparison, maize pollen was also labeled with radioactive sulfur or phosphorus, or treated with X-rays. Dominant lethality was investigated in Lilium longiflorum using pollen labeled with calcium-45, phosphorus-32 and sulfur-32. The experimental design permits one to utilize the naturally phased development of lily pollen in relation to the periods of DNA replication. If phosphorus-32 was present prior to replication, dominant lethals were produced, as detected by failure of embryo or endosperm development. Dominant lethality was less evident with calcium-45 or sulfur-35, probably due to lower amounts of isotopic incorporation into pollen. In general, the degree of response depended on the amount of isotope incorporated into the pollen (sperm). Dominant lethality was no higher than in controls when the isotope was administered after both DNA replications had been completed in maturing pollen.