Isolation by molecular cloning of a fragment of the split ovalbumin gene

Garapin, Axel-Claude; LePennec, J. P.; Roskam, Willem; Perrin, F.; Cami, B.; Krust, Andrée; Breathnach, Richard; Chambon, Pierre; Kourilsky, Philippe

doi:10.1038/273349a0

Cited by 73 publications

(37 citation statements)

References 28 publications

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“…Twelve pg of 5 Kb and 14 pg of 10 Kb DNA were ligated in a volume of 20 pl with respectively 10 pg and 5 pg of the cosmid pHC 79 DNA digested with Bam HI and used to transduce the E. Coli strain 1106, using the packaging method and strains of bacteria described by Hohn (15). Transfonned bacteria were selected by their growth on ampicillin plates and those which contained a SV40 DNA fragment were selected after in situ hybridization with 32P-labeled SV40 DNA, essentially as described by Garapin et al (16). Transformed colonies (60 plates containing about 5,000 colonies per plate) were adsorbed in nitrocellulose filters (Schleicher and Schull, BA 85) laid on the plate for 5 min.…”

Section: Materials and Methods Cellsmentioning

confidence: 99%

Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line

1981

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It has been reported that SV40-transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) produce a super T antigen of 115,000 M .This super T antigen is entirely SV40 coded and is synthesized by traRslation of an elongated form of SV40 early mRNA (May, E., Kress, M., DayaGrosjean, L., Monier, R. and May, P. (1981) J. Virol., 37,[24][25][26][27][28][29][30][31][32][33][34][35] Two recombinant cosmids were constructed by insertion of the 5 Kb and 10 Kb fragments, respectively, into cosmid pHC 79. Using restriction map analysis and nucleotide sequencing, we showed that the 5 Kb fragment actually contained the complete sequence of a gene encoding super T antigen.As compared to the normal SV40 early gene, the sequence of super T gene showed the following rearrangements: (i) The segment between nucleotides 4116 -3544 was duplicated in a direct order and (ii) these two copies of 573 nucleotide sequence were separated by a 93 nucleotide tract which was a nearly perfect inverted repeat of the segment located between nucleotides 4868 and 4776 (nucleotide numbering used here = Weissmann number + 17). INTlR)DUCIONMultiple species of T antigens with molecular weight distinct from normal sized large T antigen can be identified in a variety of SV40 transformed rat and mouse cell lines. Species of T antigen with a molecular weight considerably larger than that of normal-sized large T antigen are widespread in SV40-transformed rat or mouse cells and are referred to as super T antigen (1, 2, 3, 4).As already reported (5) SV40 transformed rat cell line V 11 F 1 clone 1 subclone 7 produces a single species of super T antigen of 115,000Mr in the absence of detectable trace of large T antigen (86,000 Mr). We have previously shown that this super T antigen (super T) is an elongated form of large T antigen, which contains a duplication of that part of large ©) IRL Prm Umited, 1 Falconberg Court, London W1V 5FG, U.K.4111

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Section: Materials and Methods Cellsmentioning

confidence: 99%

Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line

1981

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“…To form a heteroduplex between the two species, 1-3 pg/ml of each DNA were mixed together and denatured by heating for 5 min at 75°Cin 70% deionized formamide, 300 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA pH 8.5. The samples were then reannealed for 30 min at 25°C before mounting for electron microscopy as previously described (25 After centrifugation and lyophilization to remove any residual ethanol, the samples were dissolved in 30 mM NaOH, 2 mM EDTA. Electrophoresis was performed at room temperature using the alkaline gel method described by McDonnel et al (35) on 1.2% agarose alkaline gels.…”

Section: Cloningmentioning

confidence: 99%

“…23 for references). In addition we have shown that total DNA prepared from a variety of chicken tissues is indistinguishable when hybridized with purified ovalbumin probes after digestion by several restriction enzymes (24,25), with the exception of changes due to the DNA methylation pattern (26) or minor allelic variations (27,28). For a more detailed analysis, we now compare the structure of the 5'-end of the ovalbumin gene cloned from chicken erythrocyte and chicken oviduct DNA.…”

Section: Introductionmentioning

confidence: 96%

A detailed comparison of the 5′-end of the ovalbumin gene cloned from chicken oviduct and erythrocyte DNA

1980

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We have examined homologous fragments of DNA cloned from two different tissues for changes in the DNA sequence which might be related to tissue specific gene expression. The 5' end of the chicken ovalbumin gene was cloned from oviduct or erythrocyte DNA using cosmids as vectors. We have compared the two clones obtained by restriction enzyme digestions, analysis of heteroduplexes by electron microscopy or S1 nuclease digestion and by DNA sequencing. Our results show that whereas no alteration occured in the region of the gene assumed to be of importance for the control of transcription, a 4 nucleotide deletion/insertion was detected in the first intron of the ovalbumin gene.

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“…Fractionation of genomes was an intractable problem until the introduction of recombinant DNA techniques. These methods eliminate the necessity for physical separations of DNA segments and permit the isolation of structural genes from collections of randomly cloned DNA fragments (1)(2)(3)(4)(5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

“…Analysis of this material on neutral and alkaline agarose gels showed the DNA to be longer than 100 kb. DNA aliquots (20 jig) were digested in 100 Ml of 10 (20). Restriction endonucleases were prepared by published procedures (21)(22)(23)(24)(25)(26)(27) The structure of A1059 is shown in Fig.…”

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confidence: 99%

Novel bacteriophage lambda cloning vector.

Karn

Brenner

Barnett

et al. 1980

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

359

128

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A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage A vector, X1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in X1059 requires deletion of the X red and gamma genes. The recombinants are therefore spiP and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage Pg. Random fragments suitable for insertion into X1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a. This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because GA-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved X1059. By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed. DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection. Fractionation of genomes was an intractable problem until the introduction of recombinant DNA techniques. These methods eliminate the necessity for physical separations of DNA segments and permit the isolation of structural genes from collections of randomly cloned DNA fragments (1-10).Given a suitable probe, any eukaryotic gene may be isolated from a pool of cloned fragments, provided that it is large enough to give sequence representation of an entire genome. The simple multicellular eukaryote Caenorhabditis elegans has a haploid DNA content of approximately 8 X 107 base pairs (bp) (11). If random DNA cleavage and uniform cloning efficiency are assumed, a collection of 8 X 104 clones with an average length of 104 bp will be sufficient to include any genomic sequence with >99% probability. Similarly, the human genome with 2 X 109 bp will be covered by 106 clones 104 bp long.We have developed a novel bacteriophage X cloning vector, X1059, with properties that simplify the construction of recombinant phage collections representing genomes. The strategy is shown in Fig. 1 The structure of A1059 is shown in Fig. 2. The phage is a BamHI substitution vector composed of three BamHI fragments: a 19.6-kb left arm carrying the genes for the A head and tail proteins, a 17-kb central fragment, and a 9.4-kb right arm carrying the A replication and lysis genes. The two arms of the vector contain all the essential functions required for A replication and maturation in a DNA sequence 58.2% of the wildtype length. Viable phages are produced when these arms are hybridized with internal DNA fragments 12.8% to 49.8% the length of wild-type A (6.3-24.4 kb). The two arms alone do not produce viable phages, because lambdoid phages require genome sizes between 70% and 108% of the wild-type DNA to fill the phage heads properly (37, 38).The central BamHI fragment of the vector balancing the phage arms carried the A red (exo and 3 genes) and gamma functions under the control of the leftward promoter (pL) an...

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Isolation by molecular cloning of a fragment of the split ovalbumin gene

Cited by 73 publications

References 28 publications

Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line

Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line

A detailed comparison of the 5′-end of the ovalbumin gene cloned from chicken oviduct and erythrocyte DNA

Novel bacteriophage lambda cloning vector.

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