Kinetic studies of gene frequency

Harrison, Paul R.; Birnie, G.D.; Hell, Anna K.; Humphries, S. E.; Young, Bryan D.; Paul, John

doi:10.1016/0022-2836(74)90115-6

Cited by 126 publications

(51 citation statements)

References 52 publications

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“…Cytoplasmic RNA was then extracted from either total or immature erythroid cells and the globin mRNA content in these RNA preparations was determined by titration with globin cDNA. The theoretical and experimental basis of this titration method has been validated rigorously in previous papers (8,9). A constant amount of highly labeled globin cDNA was annealed under stringent conditions with increasing amounts of RNA of interest, under conditions in which hybridization of any complementary sequences present is known to be complete (9).…”

Section: Conventional Hybridization Studiesmentioning

confidence: 99%

“…cDNA prepared in this way represents a faithful but partial, transcript of a and/3 globin mRNAs (8,9).…”

Section: Preparation Of Globin C D N Amentioning

confidence: 99%

“…Hybrids were diluted in 0.5 M NaCI, 25 mM HEPES, 0.5 mM EDTA, pH 6.8, resealed in capillaries, and then heated for 5 min at various temperatures in the range 43 90°C. Capillaries were then immersed in an ice/salt mixture, expelled with S1 nuclease buffer and assayed for resistance to SI nuclease exactly as described previously (8).…”

Section: Determination O F Melting Temperaturesmentioning

confidence: 99%

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IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

Harrison

Conkie

Affara

et al. 1974

The Journal of Cell Biology

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Globin m R N A levels in 11-15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled D N A copy (cDNA) of adult globin messenger R N A s (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 1 lth day of development to 80-85% by days 13-15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin m R N A s begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition.The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin m R N A s has been confirmed by comparing the hybridization in solution of globin c D N A to cytoplasmic R N A extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.During the llth-16th day of fetal mouse development, the liver becomes the main erythropoietic organ. At this time, the fetal liver consists of up to 70% erythroid cells, comprising both multipotential and intermediate "'stem cells" and all the recognizable erythroid cell types. Although erythroid cell development represents a continuous process, certain erythroid cell types have been classified, as summarized in the scheme below [see (1, 2) for review]:

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Section: Conventional Hybridization Studiesmentioning

confidence: 99%

“…cDNA prepared in this way represents a faithful but partial, transcript of a and/3 globin mRNAs (8,9).…”

Section: Preparation Of Globin C D N Amentioning

confidence: 99%

Section: Determination O F Melting Temperaturesmentioning

confidence: 99%

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IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

Harrison

Conkie

Affara

et al. 1974

The Journal of Cell Biology

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“…On the other hand, it is known that in a DNA . cDNA reaction, the driver sequences are reacting somewhat quicker than the driver-tracer sequences, perhaps due to differences in the length of driver to tracer [19]. In any case, these results show clearly that the genes coding for calliphorin mRNA are transcribed from the unique portion of the genome, a situation similar to that found for a great number of genes in different tissues and species.…”

Section: Hybridization Between Total Dna From Calliphora and Drosophimentioning

confidence: 50%

Purification and Reverse Transcription of the Messenger RNA Coding for the Insect Protein, Calliphorin, Isolated from Larvae of the Blowfly, Calliphora vicina R.‐D.

Alonso

Flytzanis

Schätzle

et al. 1979

European Journal of Biochemistry

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Starting from poly(A)-containing RNA prepared from the fat body of larvae of the blowfly Calliphora vicina, we have purified an mRNA coding for the protein calliphorin, which is a major blowfly protein accounting for approximately 9 % of the total poly(A)-containing mRNA activity in the fat body of 5-days-old larvae, as demonstrated by translation in vitro. The mRNA for this protein was purified by sucrose gradient centrifugation under denaturing conditions and identified by cell-free translation. The peak fraction of the gradient shows three bands of about 20 S after formamide/acrylamide gel electrophoresis. Using reverse transcriptase and Calliphora mRNA isolated directly from the acrylamide gel electrophoresis or from sucrose gradients, we synthesized a cDNA probe. This cDNA hybridizes with the template, showing a major kinetic component with a molecular weight of 2.8 x lo6, fitting well with the three bands observed in the electrophoresis. Hybridization of the cDNA with total sonicated Calliphora DNA shows annealing only at very high cot values, indicating that Calliphora mRNA is transcribed from the unique portion of the fly genome.During development of Diptera larvae, a group of closely related proteins are synthesized in the fat body [l-71 and accumulate, making up at a certain stage more than half of the proteins soluble in dilute salt solutions [I, 3,5,7]. In the blowfly Calliphora vicina, a single protein has been identified, composed of six or seven identical subunits with a molecular weight of approximately 80000 each, known as calliphorin [I, 21 ; it is immunochemically related to drosophilin and luciphilin, the proteins of Drosophila [l, 5,6] It has been demonstrated that the biosynthesis of calliphorin is restricted to 3 -5-days-old larvae [7,8] ; furthermore, translatable calliphorin mRNA appears in 3-days-old larvae, reaches maximal concentrations in 4 -5-days-old animals and then decreases, being absent in pupae or imagos [7]. It is evident that the determination of the mRNA content by means of translational activity encompasses only the functionally active mRNA. We have, therefore, in order to account for all of the RNAs containing the sequences for calliphorin, proceeded to purify the calliphorin mRNA and with the help of reverse transcriptase synthesize its cDNA. The results of these experiments will be presented in this paper. MATERIALS AND METHODS Reagen AnimalsCalliphora vicina R.D. larvae were reared on beef meat at 25 "C and 65 % relative humidity. They pupate under these conditions within 7-8 days after egg deposition. Maximal synchronization was ensured by allowing deposition of eggs only for 60 min.

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“…From the published data on reassociation of mouse DNA [33,34] it can be calculated [33,10] that unique DNA of mouse under conditions of ionic strength and molecular weight used here annealed at a cot of 2-3000 mol s 1-'. The RNA/DNA hybridization with a cot '1, of 360 mol s I-' is therefore some 6-8 times faster than the reassociation of unique sequences and so the histone genes are clearly repetitive.…”

Section: Interpretation Of the Rnaidna Hybridization Kineticsmentioning

confidence: 99%

Histone-Gene Reiteration in the Genome of Mouse

Jacob

1976

Eur J Biochem

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Histone messenger RNA labelled to high specific activity has been isolated and purified from the mouse tissue-culture cell line L929. It had an electrophoretic mobility equivalent to a mean molecular weight of 1.4 x 10'. The synthesis of this RNA was suppressed, but apparently not completely so, by inhibition of cellular DNA synthesis to more than 95 %, by addition of cytosine arabinoside. The synthesis of other RNA species remained unaffected by this treatment.Poly(A)-containing mRNA, representing a 20 "/, contaminant of the electrophoretically purified histone mRNA, was removed by poly(U)-Sepharose chromatography. Histone mRNA thus purified was hybridized to DNA fractions enriched for histone genes of the sea urchin Psammechinus miliaris by actinomycin/CsCl gradient centrifugation. The mRNA was eluted from the hybrids and challenged with mouse DNA in vast DNA excess conditions. A cotll2 of 360 mol s 1-was obtained from the RNA trace curve, suggesting a 10-20-fold reiteration of the histone genes in the haploid genome. Thus histone genes in mouse are much less highly reiterated than in sea urchins, but nevertheless are present in considerable excess over the number theoretically necessary for histone synthesis during the S phase of the cell cycle.The histone DNA genes of sea urchins are so far the only certain example of protein-coding repetitive DNA sequences in the genome of eukaryotes [1,2]. It is generally held that the high degree of reiteration in sea urchins arises from the need to meet the high demand for histone proteins during an embryonic stage where cell divisions occur as frequently as every 30 min. This interpretation leads to the expectation that in other organisms with slower rates of cell division histone genes would be less reiterated, even unique, in keeping with the metabolic needs arising from the more commonly observed duration of each S phase of 6 h or more in the cell cycle.Here, the degree of reiteration of the histone genes in a mammal is reported. Mouse was chosen for this investigation since histone mRNA and its metabolism has been previously studied in detail in the mouse cell line [3 -51 and because large quantities of DNA for hybridization in vast excess can be obtained with relative ease from laboratory mice. This paper will show that the degree of reiteration of histone genes in the species is low, being approximately 10 to 20 copies per haploid chromosome set.Ahhreviations. cRNA, complementary RNA ; Cit/NaCl, 0.035 M trisodium citrate, 0.15 M sodium chloride, pH 7.0; rDNA, ribosomal DNA. MATERIALS AND METHODS MaterialsMedia for cell growth in spinner culture were from Gibco, Long Island. Fetal bovine serum and calf serum were supplied by Flow. Yeast tRNA grade 111, RNase A, pronase and cytosine arabinoside were obtained from Sigma, Before use, yeast tRNA was adsorbed to a DEAE-cellulose column, at 0.2 M sodium chloride, 20 mM sodium acetate, pH 4.5, eluted with 1 M sodium chloride, 20 mM sodium acetate, pH 4.5, shaken with phenol and precipitated with ethanol. RNase A was heat...

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Kinetic studies of gene frequency

Cited by 126 publications

References 52 publications

IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

Purification and Reverse Transcription of the Messenger RNA Coding for the Insect Protein, Calliphorin, Isolated from Larvae of the Blowfly, Calliphora vicina R.‐D.

Histone-Gene Reiteration in the Genome of Mouse

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