Use of fusions to thymidine kinase.
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3T3 Cells↗
Biomedical subjects
C Karreman
Publications and source records attributed to C Karreman.
A new set of positive/negative selectable markers for mammalian cells.
Five new positive and negative selectable markers were created for use in mammalian cells. Their negative selectabilities are based on the Thymidine kinase (Tk) gene of Herpes Simplex virus (HSV) or the Cytidine deaminase (codA) gene of E. coli. The markers can be selected positively by their ability to induce either Hygromycin (Hyg), neomycin (neo), puromycin (PAC) or Blasticidin S (BlaS) resistance. With these markers, two complete sets of markergenes are available that induce independent negative selectable phenotypes.
3T3 Cells↗
New positive/negative selectable markers for mammalian cells on the basis of Blasticidin deaminase-thymidine kinase fusions.
Two positive and negative selectable markers were created for use in mammalian cells. They are based on two genes for the resistance to Blasticidin S (BlaS) and on the thymidine kinase (Tk) gene of herpes simplex virus (HSV). The markers can be selected positively by their ability to induce BlaS resistance and negatively on the induced sensitivity towards gancyclovir (GANC). Both constructs are also expressed in Escherichia coli and transfer BlaS resistance to this organism as well, making these markers very suitable for the construction of shuttle vectors.
3T3 Cells↗
Fusion PCR, a one-step variant of the "megaprimer" method of mutagenesis.
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Artificial Gene Fusion↗
Retroviral infection of Syrian hamster BHK cells depends on age and susceptibility toward sialidase.
Infection with recombinant retroviruses is a standard method to efficiently manipulate murine or human cells genetically. Due to differences in glycosylation of the hamster homologue to the murine ecotropic receptor and the absence of an amphotropic receptor, murine retroviruses are unable to infect hamster cells. Here we describe an isolated cell clone of the Baby Hamster Kidney cell line, BHK-21, that can be successfully infected with ecotropic murine retroviruses. This finding prompted us to investigate the possibility of changing the infectability of other cell strains by manipulating the receptors of these cell lines in vitro. We found that treatment of other BHK cell clones with sialidase from C. Perfringens makes these cell clones also permissive to murine ecotropic retroviruses. Long term cultivation of three tested cell clones increased this susceptibility. Although the resulting infectants show a higher rate of infectability, they still require the manipulation with sialidase for this superinfection to be efficient.
3T3 Cells↗
On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines.
A pilot experiment for the construction of a hamster derived, high producer cell line using site specific recombination is described. In the experiment chromosomal loci with intrinsic high expression characteristics were sought via infection with a retroviral construct, containing double FRT sites and subsequent screening for overproduction of an encoded markergene. These sites were then targeted with a second vector, that recombined via the FLP/FRT system from Saccharomyces cerevisiae yielding cells that had the second construct at exactly the same position as the first. By using retroviral vectors with double and single FRT sites, respectively, stable clones can be created that can no longer be excised with FLP.
Animals↗
A dotplot program for the Atari ST, for the analysis of DNA and protein sequences.
A program was written in GFA-BASIC for the Atari ST microcomputer aimed at drawing two-dimensional homology 'dotplot' patterns for two protein or DNA sequences. The program, built around a machine-code subroutine, communicates interactively with the user by means of a multi-button dialogue panel and mouse-directed input. A 1000 X 1000 sequence comparison with a 14:21 stringency window takes 12 s.
Algorithms↗
Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle.
Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.
Amino Acid Sequence↗
Agmenellum quadruplicatum M.AquI, a novel modification methylase.
The complete type II modification methylase of Agmenellum quadruplicatum was cloned in Escherichia coli as an R.Sau3A fragment of approximately 4.5 kilobases. The coding sequence was contained in a stretch of 1,156 base pairs which was organized into two parallel, partly overlapping open reading frames of 248 and 139 codons. In vivo complementation experiments showed that the synthesis of both predicted peptides was required for full methylase activity. The amino acid sequences were considerably similar to regions of other deoxycytidylate methylases.
Amino Acid Sequence↗
Cloning and complete nucleotide sequences of the type II restriction-modification genes of Salmonella infantis.
The complete type II restriction-modification system of Salmonella infantis was cloned in Escherichia coli as an R . Sau3AI fragment of 3,430 base pairs. The clone was shown to express the restriction endonuclease as well as the modification methylase. The nucleotide sequence of the above fragment showed two open reading frames of 461 and 230 codons in tail-to-tail orientation. These were shown to represent the modification methylase M . SinI and the restriction endonuclease R . SinI, respectively. The methylase M . SinI amino acid sequence revealed a considerable similarity to those of other deoxycytidylate methylases. In contrast, endonuclease R . SinI did not exhibit such a similarity to other restriction enzymes.
Amino Acids↗
Isolation and characterization of the modification methylase M . SinI.
A sequence-specific modification methylase (M . SinI) was isolated and purified from Escherichia coli harboring a derivative of recombinant plasmid pSI4 (see accompanying manuscript: C. Karreman and A. de Waard, J. Bacteriol. 170:2527-2532, 1988), which contains a Salmonella infantis DNA insert. The enzyme uniquely methylates the internal deoxycytidylate residue in the nucleotide sequence GG(A/T)MeCC, thereby protecting DNA completely against cleavage by restriction endonuclease R . SinI or R . AvaII [GG(A/T)CC], and in part against cleavage by R . Sau96I (GGNCC).
Base Sequence↗
Isolation of a deoxycytidylate methyl transferase capable of protecting DNA uniquely against cleavage by endonuclease R.Aqu I (isoschizomer of Ava I).
A sequence-specific modification methylase (M.AquI) was isolated and purified from Agmenellum quadruplicatum (Synechococcus PCC 7002). This enzyme uniquely methylates the deoxycytidylate residue in the sequence *CYCGRG indicated by the asterisk. It was shown to protect DNA against cleavage by restriction endonucleases AvaI, SmaI and XhoI, which recognize the sequences CYCGRG, CCCGGG, and CTCGAG, respectively.
Base Sequence↗
Ner, a cro-like function of bacteriophage Mu.
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Bacteriophage mu↗