[68] Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease S1 gel mapping

Favaloro, Jenny M.; Treisman, Richard; Kamen, Robert

doi:10.1016/s0076-6879(80)65070-8

Cited by 1,351 publications

(538 citation statements)

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“…Cytoplasmic RNA was purified from erythrocytes of N. coriiceps (14,15), and poly(A)+ RNA was selected by affinity chromatography on oligo(dT)-cellulose (16). (dT)12_18-primed, double-stranded cDNA was synthesized from the poly(A)+RNA (17), the cDNA was ligated by means of EcoRI adapters into the phagemid pT7T3 18U (Pharmacia), and a library was generated by electroporation-mediated transformation (18) of competent Escherichia coli NM522 cells with the recombinant phagemid stock.…”

Section: Methodsmentioning

confidence: 99%

Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

Cocca

Ratnayake-Lecamwasam

Parker

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

154

107

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Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using a-and f-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globinrelated DNA sequences. We detect specific, high-stringency hybridization of the a-globin probe to genomic DNAs of both white-and red-blooded species, whereas the 8-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of a-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes p-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult ,3-globin locus prior to the diversification of the clade.In 1954 Ruud (1) published the first systematic analysis of the "white" blood of an antarctic icefish, Chaenocephalus aceratus. He reported that fresh blood was nearly transparent, contained leukocytes at 1% by volume, but lacked erythrocytes and the respiratory transport pigment hemoglobin. Furthermore, the oxygen-carrying capacity of C. aceratus blood was approximately 10% that of two red-blooded notothenioids. Subsequent investigations extended these observations to other icefish species and revealed that icefish blood contains small numbers of "erythrocyte-like" cells that, nevertheless, are devoid of hemoglobin (2, 3). Thus, limited to oxygen physically dissolved in their blood, the icefishes have evolved compensatory physiological and circulatory adaptations-e.g., modest suppression of metabolic rates, enhanced gas exchange by large, well-perfused gills and cutaneous respiration, and large increases in cardiac output and blood volume-that ensure adequate oxygenation of their tissues (4, 5).We have initiated studies to determine the status of globin genes in channichthyid genomes and to evaluate potential evolutionary mechanisms leading to the hemoglobinless phenotype. Icefishes evolved from the red-blooded Notothenioidei (6, 7), which, in contrast to temperate fishes, are characterized by a paucity of hemoglobin forms (7-10). Adults of the family Nototheniidae (antarctic rockcods) generally possess a major hemoglobin, Hb 1 (-95% of the total), and a second, minor hemoglobin, Hb 2, that differ in their a chains (al and a2, respectively) (11-13). The more phyletically derived harpagiferids and bathydraconids have a single hemoglobin. The trend toward reduced hemoglobin multiplicity in t...

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Section: Methodsmentioning

confidence: 99%

Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

Cocca

Ratnayake-Lecamwasam

Parker

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

154

107

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“…Total RNA, poly(A)+ mRNA, and cytoplasmic and nuclear RNAs were prepared by the standard protocols (12,13). RNA gel blot transfer and hybridization were carried out as described by Maniatis et al (13).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional control of the endogenous MYC protooncogene by antisense RNA.

Yokoyama

Imamoto

1987

Proc. Natl. Acad. Sci. U.S.A.

129

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DNAs (20 ,ug) by the protoplast fusion method (10). Two days later, selection for the ability to grow in Dulbecco's modified Eagle's medium (DMEM) containing mycophenolic acid (25 ,gg/ml), aminopterin, xanthine, hypoxanthine, and thymidine was performed as described by Mulligan and Berg (11). Transformants resistant to a high dose of mycophenolic acid (110 Ag/ml) were obtained by culturing cells in DMEM plus mycophenolic acid over a period of 6 months during which the mycophenolic acid concentration was increased at 2-week intervals. Secondary and tertiary transformants were obtained by retransfecting a primary and a secondary transformant, respectively, with plasmid DNA (10 ,ug/ml) followed by culturing in DMEM containing mycophenolic acid (110 ,g/ml). Control antisense and sense clones (pSV2gpt+, pSVgptC5-8, pSVH-2Kb, and pSVa-globin) were obtained by the cotransfection with pSV2gpt+. The same amplification protocol and the repeated transfection were used as described above.RNA Isolation and Analysis. Total RNA, poly(A)+ mRNA, and cytoplasmic and nuclear RNAs were prepared by the standard protocols (12, 13). RNA gel blot transfer and hybridization were carried out as described by Maniatis et al.(13). [a-32P]dCTP-labeled single-stranded probes were prepared from restriction fragments containing f,-actin cDNA (14) or exons 2 and 3 of MYC cDNA by exonuclease III digestion, reverse transcriptase treatment (15), and strand separation (13 7363The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…LA1-55n cells were lysed with NP-40 bu er (Favaloro et al, 1980) and centrifuged at 2500 g for 5 min. The supernate was used as cytoplasmic fraction.…”

Section: Cell Fractionationmentioning

confidence: 99%

HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts

et al. 1999

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N-myc gene copy numbers and transcription rates are similar in N (neuroblastic, tumorigenic) and S (nonneuronal, non-tumorigenic) neuroblastoma cells with chromosomally integrated ampli®ed N-myc genes. However, N cells show signi®cantly higher N-myc mRNA levels than S cells. Therefore, post-transcriptional control of N-myc gene expression must di er between these cell types. Since no di erences in N-myc mRNA half-life were found between N and S cells from two cell lines, steady-state levels of N-myc pre-mRNA processing intermediates were analysed. Results suggest that the di erences in N-myc expression arise primarily at the nuclear post-transcriptional level. The neuronal-speci®c RNA-binding Hu proteins are present in cytoplasmic and nuclear fractions of N cells and one of them, HuD, binds speci®cally to both exonic and intronic N-myc RNA sequences. In sense and antisense HuD-transfected N cells, there are coordinate changes in HuD and N-myc expression levels. Thus, we propose that HuD plays a role in the nuclear processing/stability of N-myc premRNA in N-type neuroblastoma cells.

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[68] Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease S1 gel mapping

Cited by 1,351 publications

References 30 publications

Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

Transcriptional control of the endogenous MYC protooncogene by antisense RNA.

HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts

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