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At least 199 records · Page 11Linked to original sources
PubMed1975

X-ray- and TEM-induced mitotic recombination in Drosophila melanogaster: unequal and sister-strand recombination.

Twin mosaic spots of dark-apricot and light-apricot ommatidia were found in the eyes of wa/wa females, of wa males, of females homozygous for In(1)sc4, wa and of attached-X females homozygous for wa. The flies were raised from larvae which had been treated with 1630 R of X-rays at the age of 48-52 hours. An additional group of wa/wa females and wa males came from larvae that had been fed with triethylene melamine (TEM) at the age of 22-24 hours. The twin spots apparently were the result of induced unequal mitotic recombination, i.e. from unequal sister-strand recombination in the males and from unequal sister-strand recombination as well as, possibly, unequal recombination between homologous strands in the females. That is, a duplication resulted in waDp wa/wa dark-apricot ommatidia and the corresponding deficiency in an adjacent area of wa/Df wa light-apricot ommatidia. In an additional experiment sister-strand mitotic recombination in the ring-X chromosome of ring-X/rod-X females heterozygous for w and wco is believed to be the cause for X-ray induced single mosaic spots that show the phenotype of the rod-X marker.

Drosophila melanogaster↗
PubMed1984

Enhanced recombination between lambda plac5 and mini-F-lac: the tra regulon is required for recombination enhancement.

F42lac recombination with lambda plac is normally 20-fold to 50-fold higher than recombination between lambda plac and a chromosomal lac gene. Transductional crosses with lambda plac and a recombinant plasmid containing the lac operon and F replication functions show only two to four-fold higher recombination than similar crosses with a chromosomal lac gene. Insertion of a BamHI fragment containing the entire tra regulon of F into the mini-F-lac plasmid restores the high level of recombination seen with F42lac.

Bacteriophage lambda↗
PubMed1976

Recombined molecules of mitochondrial DNA obtained from crosses between cytoplasmic petite mutants of Saccharomyces cerevisiae: the stoichiometry of parental DNA repeats within the recombined molecule.

We have studied recombination between repetitive mitochondrial DNAs from cytoplasmic petite mutants of Saccharomyces cerevisiae. Mitochondrial DNA was isolated from two parental p- mutants, carrying respectively the CR and the ER mitochondrial genetic markers, and from two p- CRER diploid genetic recombinants. These two recombinants, obtained from the same parental petites, differ in their degrees of suppressiveness. The p- mitochondrial DNAs were analyzed by DNA-DNA hybridization, high resolution melting and reassociation kinetics. It was found that the repeating unit of the CR parental p- DNA is 3 to 4 times longer than that of the ER parent. There is very little sequence homology between these two p- mitochondrial DNAs and almost all parental sequences are integrated into the recombined molecules. Mitochondrial DNA from both types of recombinants seems to contain the two parental repeating units in the ratio 1:1.

Base Sequence↗
PubMed1983

Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination.

Bacteriophage P1 encodes its own site-specific recombination system consisting of a site at which recombination takes place called loxP and a recombinase called Cre. A number of lambda and plasmid substrates containing two loxP sites have been constructed. Using these substrates we have shown both in vivo and in vitro that a fully functional loxP site is composed of no more than 60 bp. In vitro, when an extract containing Cre is used, recombination between loxP sites on supercoiled, nicked-circle or linear DNA occurs efficiently. The most surprising result from the in vitro studies is that 50% of the products of recombination between loxP sites on a supercoiled DNA substrate are present as free supercoiled circles. The ability to produce free products starting with a supercoiled substrate suggests a rather unique property of Cre-mediated lox recombination, the implications of which are discussed in terms of possible effects of the protein on the topology of the DNA molecule.

Bacteriophage lambda↗
PubMed1995

Purification of recombinant human coagulation factors II and IX and protein S expressed in recombinant Vaccinia virus-infected Vero cells.

Recombinant human coagulation factors II and IX and recombinant human protein S were expressed by the Vaccinia virus expression system in Vero cells and isolated from the serum-free media. All three recombinant proteins were purified by the same universal strategy. After concentration by anion exchange chromatography, recombinant coagulation factors were purified by anion exchange filtration in the presence of Ca2+. The addition of Ca2+ to the vitamin K-dependent coagulation factors modified their physico-chemical behavior during anion exchange chromatography and resulted in the separation of the recombinant coagulation proteins from contaminating proteins.

Animals↗
PubMed1995

Over-expression of human sex steroid-binding protein (hSBP/hABP or hSHBG) in insect cells infected with a recombinant baculovirus. Characterization of the recombinant protein and comparison to the plasma protein.

Human sex steroid-binding protein (hSBP/hABP or hSHBG) was over-expressed in High Five and Sf9 cells adhered to plates and in suspension. The adherent cells expressed to levels of 2.3 mg/l and 1.4 mg/l after 4 and 6 days, respectively, while Sf9 cells grown in suspension yielded 4.67 mg/l after 6 days. Recombinant hSBP/hABP, purified to homogeneity by immunoadsorption, was found to fold similarly to native plasma hSBP/hABP and to display similar sequence epitopes after heat denaturation. The recombinant protein binds dihydrotestosterone, testosterone, and 17 beta-estradiol with KdS of 0.6, 2.4, and 14.2 nM, respectively, which are similar to plasma hSBP/hABP. The recombinant protein contains N-linked and O-linked oligosaccharide side-chains but the monomer exhibits a slightly lower molecular weight than plasma hSBP/hABP (40 kDa vs 44 kDa) which may be due to the absence of one N-linked side-chain or to shorter oligosaccharide side-chains. The partial N-terminal sequence LRPVLP(T)Q of recombinant hSBP/hABP is identical to plasma hSBP/hABP but appears to be less heterogeneous. These results indicate that recombinant baculovirus SBP represents a good model for investigating the structure of plasma hSBP/hABP. The expression system will allow the isolation of preparative amounts of SBP mutants generated by combinatorial site-directed mutagenesis to advance investigations on structure-function relationships and undertake crystallization trials for X-ray diffraction analyses.

Amino Acid Sequence↗
PubMed2005

Amended recombinant cells (ARCs): an economical and surprisingly effective production and delivery vehicle for recombinant bovine IFN-gamma.

Recombinant Pseudomonas fluorescens cells, expressing over 40% protein as bovine interferon-gamma (IFN-gamma), were chemically fixed to sterilize the culture and amend the bacterial cell wall. When killed and fixed recombinant cells, termed here amended-recombinant-cells (ARCs), were assayed for interferon activity, we obtained the following surprising results: 1) sterilization and fixation did not inactivate ARC-encapsulated IFN-gamma; 2) ARC-encapsulated IFN-gamma and soluble, recombinant IFN-gamma were equally active in vitro but proteolysis was required for release of the ARC cytokine; and 3) ARC-encapsulated IFN-gamma was active in vivo with optimal adjuvant activity at a dose about 1000-fold less than previously reported for soluble, recombinant IFN-gamma and 100-fold less than doses which induced adverse systemic effects. The mechanism by which ARC-encapsulation increased IFN-gamma activity in vivo remains uncertain. However, our in vitro results show that sustained release of soluble IFN-gamma is a likely factor. The ARC production and delivery system achieves enhanced adjuvant activity with reduced risk of systemic effects, and the low cost of IFN-gamma production offers new opportunities for the use of this important cytokine.

Animals↗
PubMed2005

Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.

V(D)J recombination is the process by which the variable region exons encoding the antigen recognition sites of receptors expressed on B and T lymphocytes are generated during early development via somatic assembly of component gene segments. In response to antigen, somatic hypermutation (SHM) and class switch recombination (CSR) induce further modifications of immunoglobulin genes in B cells. CSR changes the IgH constant region for an alternate set that confers distinct antibody effector functions. SHM introduces mutations, at a high rate, into variable region exons, ultimately allowing affinity maturation. All of these genomic alteration processes require tight regulatory control mechanisms, both to ensure development of a normal immune system and to prevent potentially oncogenic processes, such as translocations, caused by errors in the recombination/mutation processes. In this regard, transcription of substrate sequences plays a significant role in target specificity, and transcription is mechanistically coupled to CSR and SHM. However, there are many mechanistic differences in these reactions. V(D)J recombination proceeds via precise DNA cleavage initiated by the RAG proteins at short conserved signal sequences, whereas CSR and SHM are initiated over large target regions via activation-induced cytidine deaminase (AID)-mediated DNA deamination of transcribed target DNA. Yet, new evidence suggests that AID cofactors may help provide an additional layer of specificity for both SHM and CSR. Whereas repair of RAG-induced double-strand breaks (DSBs) involves the general nonhomologous end-joining DNA repair pathway, and CSR also depends on at least some of these factors, CSR requires induction of certain general DSB response factors, whereas V(D)J recombination does not. In this review, we compare and contrast V(D)J recombination and CSR, with particular emphasis on the role of the initiating enzymes and DNA repair proteins in these processes.

Animals↗
PubMed2004

SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast.

It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

Cell Cycle Proteins↗
PubMed2000

Initiation of genetic recombination and recombination-dependent replication.

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.

DNA Damage↗
PubMed1992

Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.

Aequorin is a calcium-dependent bioluminescent protein isolated from the hydromedusan Aequorea victoria. The gene for aequorin has been cloned and overexpressed in Escherichia coli [Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126, 1259; Prasher et al. (1987) Biochemistry 26, 1326]. Higher levels of expression have recently been obtained by subcloning aequorin cDNA into the pRC23 plasmid vector such that its expression is under control of the lambda PL promoter [Cormier et al. (1989) Photochem. Photobiol. 49, 509]. Purification of recombinant apoaequorin from E. coli containing this new recombinant plasmid (pAEQ1.3) was accomplished by a two-step procedure involving gel filtration and anion-exchange chromatography on Sephadex G-100 and DEAE-Sepharose, respectively. Typically, 400-500 mg of recombinant protein was obtained from 100 L of fermentation culture. The purified recombinant apoaequorin could be converted to aequorin in high yield upon incubation with synthetic coelenterate luciferin, dissolved oxygen, and a thiol reagent with a photon yield similar to the native photoprotein. Detection of recombinant aequorin in the Dynatech ML1000 Microplate luminometer was linear between 10(-18) and 10(-12) mol, and little loss of specific activity was observed when the protein was derivatized with biotin. The biotinylated derivative was stable when frozen, lyophilized, or stored at 4 degrees C. The feasibility of using biotinylated aequorin as a nonradioactive tag was established by its application in a variety of solid-phase assay formats using the high-affinity streptavidin/biotin interaction. A microtiter-based bioluminescent immunoassay (BLIA) using biotinylated aequorin and the ML1000 luminometer was developed for the detection of subnanogram amounts of a glycosphingolipid (Forsmann antigen). In addition, nanogram to subnanogram quantities of protein antigens and DNA, immobilized on Western and Southern blots, respectively, were detected on instant and X-ray films using biotinylated aequorin.

Aequorin↗
PubMed1993

Preparation of active recombinant TIMP-1 from Escherichia coli inclusion bodies and complex formation with the recombinant catalytic domain of PMNL-collagenase.

TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The Ki values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus.

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.

Adenoviridae↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed1978

Time of recombination in the Drosophila melanogaster oocyte: evidence from a temperature-sensitive recombination-deficient mutant.

A temperature-sensitive recombination-deficient mutant, rec-126, has been isolated that permits high frequencies of recombination at the permissive temperature (25 degrees) but greatly decreases recombination at the restrictive temperature (31 degrees). The sensitive period for response of female germ cells carrying this mutant to the restrictive temperature has been defined. Sensitivity begins very close to the time the oocyte enters premeiotic interphase and initiates DNA synthesis; it continues for the duration of premeiotic-S; and it terminates with the completion of S. This time span precisely coincides with the sensitive period for enhancement of recombination by heat in the normal genome and is further characterized by the presence of the synaptonemal complex. These results provide compelling evidence for identifying premeiotic-S as the time of meiotic recombination.

Animals↗
PubMed1991

Mouse kappa light-chain recombination signal sequences mediate recombination more frequently than do those of lambda light chain.

Immunoglobulin and T-cell receptor genes are somatically rearranged by site-specific recombination. Recombination signal sequences (RSS) have been identified as the major targeting element of this process. Recent reports demonstrate that differences in RSS affect the frequency of recombination, suggesting a role for RSS in the development of the B-cell repertoire. Examination of mouse light-chain RSS indicates that kappa light-chain RSS consistently show a greater degree of similarity to a consensus sequence than do those of lambda light chain. To determine whether this difference in natural RSS could affect the patterns of light-chain gene rearrangement and expression, we have constructed recombination substrates containing both a typical mouse kappa RSS pair and a typical mouse lambda RSS pair. Experiments using these substrates demonstrate that the kappa RSS pair mediates recombination at a vastly higher frequency than does the lambda RSS pair. This result argues that RSS differences may contribute significantly to the patterns of mouse immunoglobulin light-chain rearrangement, ultimately resulting in a high proportion of kappa light chain relative to lambda.

Animals↗
PubMed2006

Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences.

The shufflon of plasmid R64 consists of four DNA segments separated and flanked by seven sfx recombination sites. Rci-mediated recombination between any inverted sfx sequences causes inversion of the DNA segments independently or in groups. The R64 shufflon selects one of seven pilV genes encoding type IV pilus adhesins, in which the N-terminal region is constant, while the C-terminal regions are variable. The R64 sfx sequences are asymmetric. The sfx central region and right arm sequences are conserved, but left arm sequences are not. Here we constructed a symmetric sfx sequence, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence and made artificial shufflon segments carrying symmetric sfx sequences in inverted or direct orientations. The symmetric sfx sequence exhibited the highest inversion frequency in a shufflon segment flanked by two inverted sfx sequences. Rci-dependent deletion of a shufflon segment flanked by two direct symmetric sfx sequences was observed, suggesting that asymmetry of R64 sfx sequences inhibits recombination between direct sfx sequences. In addition, intermolecular recombination between symmetric sfx sequences was also observed. The extra C-terminal domain of Rci was shown to be essential for inversion of the R64 shufflon using asymmetric sfx sequences but not essential for recombination using symmetric sfx sequences, suggesting that the Rci C-terminal segment helps the binding of Rci to asymmetric sfx sequences. Rci protein lacking the C-terminal domain bound to both arms of symmetric sfx sequence but only to the right arm of asymmetric sfx sequence.

Base Sequence↗
PubMed2005

A modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints.

We have developed a modified BOOTSCAN algorithm that may be used to screen nucleotide sequence alignments for evidence of recombination without prior identification of nonrecombinant reference sequences. The algorithm is fast and includes a Bonferroni corrected statistical test of recombination to circumvent the multiple testing problems encountered when using the BOOTSCAN method to explore alignments for evidence of recombination. Using both simulated and real datasets we demonstrate that the modified algorithm is more powerful than other phylogenetic recombination detection methods and performs almost as well as one of the best substitution distribution recombination detection methods.

Algorithms↗