Isolation of cDNA clones for rabbit red cell carbonic anhydrase and catalase: A pilot study directed at isolation of coordinately expressed genes

S. H. Boyer, Harry Ostrer, K. D. Smith

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3 Citations (Scopus)

Abstract

Among the fraction of all genes expressed in red cells, there are a number of subsets that are candidates for dissection of the events and mechanisms underlying coordinate gene expression. Examples of such subsets include first, the α- and β-globin genes whose transcription is ordinarily adjusted so as to lead to α2β2 hemoglobin tetramers without an appreciable remainder of either α- or β-globin polypeptides; second, the ensemble of genes for the heme biosynthesis enzymes that must be coordinately expressed in conjunction with those for heme-bearing proteins such as globin and catalase; third, the genes responsible for the array of cytoskeletal proteins whose activation must perforce be correlated during creation of red cell architecture, and fourth, the genes for humans carbonic anhydrase I (CA I) and β-globin whose products exhibit parallel 100-fold increases in abundance during the first 18 months of life. We have speculated that coordinate expression of the member genes in each of these subsets is in part dependent on shared DNA sequences in and around the gene regions of each subset member. As a corollary, we suppose that these sequences will not be shared by genes that are not expressed coordinately with the subset. We thus imagine, for example, that they are absent in the carbonic anhydrase III (CA III) gene region since this enzyme is not appreciably expressed in red cells. We employed CA I and catalase as test cases to assess the feasibility of first, purifying low-abundance mRNA via immunoprecipitation of specific nascent polypeptides attached to rabbit reticulocyte polyribosomes; and second, using this purified mRNA to identify homologous clones within cDNA libraries prepared from rabbit reticulocytes. As we shall outline, both tests of feasibility were passed in each case. Specificially, methods were developed so that mRNA for carbonic anhydrase and for catalase could be obtained with 50-85% purity. Aliquots of these purified preparations, when labeled, were used to identify corresponding cDNA clones. In turn, insert DNA derived from these clones was shown to react specifically with its corresponding mRNA. It thus seems likely that the way is open for the isolation of cDNA clones for other trace entities within coordinately expressed subsets of genes. To support this belief, we now find that rabbit reticulocyte spectrin-mRNA can be enriched to a level of 2% by immunopurification of polyribosomes.

Original languageEnglish (US)
Pages (from-to)324-331
Number of pages8
JournalAnnals of the New York Academy of Sciences
VolumeVOL. 429
StatePublished - 1984
Externally publishedYes

Fingerprint

Carbonic Anhydrases
Catalase
Complementary DNA
Clone Cells
Genes
Cells
Rabbits
Globins
Reticulocytes
Messenger RNA
Carbonic Anhydrase I
Polyribosomes
Heme
Carbonic Anhydrase III
Bearings (structural)
Gene Expression
Clone
Gene
Isolation
Rabbit

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

@article{bad896366ada4a7da7ffe0e5abf8655a,
title = "Isolation of cDNA clones for rabbit red cell carbonic anhydrase and catalase: A pilot study directed at isolation of coordinately expressed genes",
abstract = "Among the fraction of all genes expressed in red cells, there are a number of subsets that are candidates for dissection of the events and mechanisms underlying coordinate gene expression. Examples of such subsets include first, the α- and β-globin genes whose transcription is ordinarily adjusted so as to lead to α2β2 hemoglobin tetramers without an appreciable remainder of either α- or β-globin polypeptides; second, the ensemble of genes for the heme biosynthesis enzymes that must be coordinately expressed in conjunction with those for heme-bearing proteins such as globin and catalase; third, the genes responsible for the array of cytoskeletal proteins whose activation must perforce be correlated during creation of red cell architecture, and fourth, the genes for humans carbonic anhydrase I (CA I) and β-globin whose products exhibit parallel 100-fold increases in abundance during the first 18 months of life. We have speculated that coordinate expression of the member genes in each of these subsets is in part dependent on shared DNA sequences in and around the gene regions of each subset member. As a corollary, we suppose that these sequences will not be shared by genes that are not expressed coordinately with the subset. We thus imagine, for example, that they are absent in the carbonic anhydrase III (CA III) gene region since this enzyme is not appreciably expressed in red cells. We employed CA I and catalase as test cases to assess the feasibility of first, purifying low-abundance mRNA via immunoprecipitation of specific nascent polypeptides attached to rabbit reticulocyte polyribosomes; and second, using this purified mRNA to identify homologous clones within cDNA libraries prepared from rabbit reticulocytes. As we shall outline, both tests of feasibility were passed in each case. Specificially, methods were developed so that mRNA for carbonic anhydrase and for catalase could be obtained with 50-85{\%} purity. Aliquots of these purified preparations, when labeled, were used to identify corresponding cDNA clones. In turn, insert DNA derived from these clones was shown to react specifically with its corresponding mRNA. It thus seems likely that the way is open for the isolation of cDNA clones for other trace entities within coordinately expressed subsets of genes. To support this belief, we now find that rabbit reticulocyte spectrin-mRNA can be enriched to a level of 2{\%} by immunopurification of polyribosomes.",
author = "Boyer, {S. H.} and Harry Ostrer and Smith, {K. D.}",
year = "1984",
language = "English (US)",
volume = "VOL. 429",
pages = "324--331",
journal = "Annals of the New York Academy of Sciences",
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TY - JOUR

T1 - Isolation of cDNA clones for rabbit red cell carbonic anhydrase and catalase

T2 - A pilot study directed at isolation of coordinately expressed genes

AU - Boyer, S. H.

AU - Ostrer, Harry

AU - Smith, K. D.

PY - 1984

Y1 - 1984

N2 - Among the fraction of all genes expressed in red cells, there are a number of subsets that are candidates for dissection of the events and mechanisms underlying coordinate gene expression. Examples of such subsets include first, the α- and β-globin genes whose transcription is ordinarily adjusted so as to lead to α2β2 hemoglobin tetramers without an appreciable remainder of either α- or β-globin polypeptides; second, the ensemble of genes for the heme biosynthesis enzymes that must be coordinately expressed in conjunction with those for heme-bearing proteins such as globin and catalase; third, the genes responsible for the array of cytoskeletal proteins whose activation must perforce be correlated during creation of red cell architecture, and fourth, the genes for humans carbonic anhydrase I (CA I) and β-globin whose products exhibit parallel 100-fold increases in abundance during the first 18 months of life. We have speculated that coordinate expression of the member genes in each of these subsets is in part dependent on shared DNA sequences in and around the gene regions of each subset member. As a corollary, we suppose that these sequences will not be shared by genes that are not expressed coordinately with the subset. We thus imagine, for example, that they are absent in the carbonic anhydrase III (CA III) gene region since this enzyme is not appreciably expressed in red cells. We employed CA I and catalase as test cases to assess the feasibility of first, purifying low-abundance mRNA via immunoprecipitation of specific nascent polypeptides attached to rabbit reticulocyte polyribosomes; and second, using this purified mRNA to identify homologous clones within cDNA libraries prepared from rabbit reticulocytes. As we shall outline, both tests of feasibility were passed in each case. Specificially, methods were developed so that mRNA for carbonic anhydrase and for catalase could be obtained with 50-85% purity. Aliquots of these purified preparations, when labeled, were used to identify corresponding cDNA clones. In turn, insert DNA derived from these clones was shown to react specifically with its corresponding mRNA. It thus seems likely that the way is open for the isolation of cDNA clones for other trace entities within coordinately expressed subsets of genes. To support this belief, we now find that rabbit reticulocyte spectrin-mRNA can be enriched to a level of 2% by immunopurification of polyribosomes.

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