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At least 19 recordsLinked to original sources
PubMed1990

Effect of recombinant interleukin-1 alpha, recombinant interleukin-2, recombinant interferon-beta, and recombinant tumor necrosis factor on subcutaneously implanted adenocarcinoma 755 and Lewis lung carcinoma.

The antitumor activity of recombinant human interleukin-1 alpha (rHIL-1 alpha), recombinant human tumor necrosis factor, and recombinant murine interferon-beta (rIFN-beta) was augmented by concomitant administration of recombinant human interleukin-2 (rHIL-2) in the treatment of adenocarcinoma 755. Especially when a divided dose (two doses/day) of rHIL-1 alpha or rIFN-beta was combined with a divided dose of rHIL-2, the antitumor effect was markedly potentiated. However, only a marginal effect was seen by combination of rHIL-1 alpha and/or rIFN-beta and rHIL-2 against Lewis lung carcinoma, a tumor that is resistant to cytokines.

Adenocarcinoma↗
PubMedDate not supplied

The RecE recombination pathway mediates recombination between partially homologous DNA sequences: structural analysis of recombination products.

Escherichia coli generalized recombination, utilizing the RecA RecB recombination pathway, requires large stretches (70-200 bp) of complete DNA sequence homology. In contrast, we have found that the RecE pathway can promote recombination between DNA with only short stretches of homology. A plasmid containing 10 partially homologous direct repeats was linearized by digestion with specific restriction enzymes. After transformation, a RecE+ (sbcA) host was able to circularize the plasmid by recombination between partially homologous direct repeat sequences. Recombination occurred in regions of as little as 6 bp of perfect homology. Recombination was enhanced in the regions adjacent to restriction sites used to linearize the plasmid, consistent with a role of double-strand breaks in promoting recombination. A mechanism is proposed in which the 5' exonuclease, ExoVIII, produces 3' single-stranded ends from the linearized plasmid. These pair with other sequences of partial homology. Partial homologies in the sequences flanking the actual join serve to stabilize this recombination intermediate. Recombination is completed by a process of "copy and join." This recombination mechanism requires less homology to stabilize intermediates than the degree of homology needed for mechanisms involving strand invasion. Its role in nature may be to increase genomic diversity, for example, by enhancing recombination between bacteriophages and regions of the bacterial chromosome.

Base Sequence↗
PubMed1985

Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer.

The alternate expression of flagellin genes in Salmonella is the result of an inversion of a 996 bp segment of chromosomal DNA. We have analyzed the components of this site-specific recombination reaction in an in vitro system derived from E. coli. Efficient Hin-mediated inversion requires the 20,000 MW Hin protein and a proteinase K-sensitive host component. The supercoiled DNA substrate must contain two 26 bp recombination sites in inverted configuration and a 60 bp sequence that increases the rate of recombination over 20-fold. This recombinational enhancer can function at many different locations and consists of at least two noncontiguous sequence domains whose relative orientation, but not precise spacing, with respect to each other is important. Synthetically derived wild-type and mutant recombination sites were constructed to analyze the sequence and structural features that are important within the recombination site.

Bacterial Proteins↗
PubMed1986

Effects of recombinant human tumor necrosis factor alpha, recombinant human gamma-interferon, and prostaglandin E on colony formation of human hematopoietic progenitor cells stimulated by natural human pluripotent colony-stimulating factor, pluripoietin alpha, and recombinant erythropoietin in serum-free cultures.

The influences of pure human pluripotent colony-stimulating factor, highly purified pluripoietin alpha, pure recombinant human tumor necrosis factor alpha, pure recombinant human gamma-interferon, and natural prostaglandin E1 (PGE1) were evaluated on colony formation of multipotential and erythroid progenitor cells in the presence of recombinant erythropoietin and hemin and on colony formation of granulocyte-macrophage progenitors in normal human marrow cultured in the presence or absence of serum. Serum was replaced by bovine serum albumin, iron-saturated transferrin, cholesterol, and calcium chloride. Increasing concentrations of pluripotent colony-stimulating factor and pluripoietin alpha stimulated increasing numbers of colonies from nonadherent low-density T-lymphocyte-depleted cells in the absence and presence of serum. Growth was usually greater in the presence of serum and on a unit basis pluripoietin alpha was more active than pluripotent colony-stimulating factor. Recombinant human tumor necrosis factor alpha and recombinant human gamma-interferon suppressed colony formation colony forming unit-granulocyte-macrophage, burst forming unit-erythroid, and colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte; PGE1 suppressed colony formation by colony-forming unit-granulocyte-macrophage, stimulated colony formation by burst forming unit-erythroid, and had no effects on colony formation by colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte in both serum-containing and serum-free medium. The PGE1 enhancing effects on erythroid colony formation required T-lymphocytes. Thus, results are similar using serum-containing and serum-free cultures of human bone marrow cells and serum-free defined culture medium can be used to study the mechanism of action of purified natural and recombinant growth and suppressor molecules in vitro.

Bone Marrow Cells↗
PubMed1990

Antitumor effect of combination of murine recombinant interferon beta, murine recombinant interferon gamma and human recombinant interleukin-2 in MethA-bearing mice.

We have previously reported that the combination of murine recombinant interferon beta (Mu-rIFN beta) with murine recombinant interferon gamma (Mu-rIFN gamma) provided greater inhibition of tumor growth than did each one alone in MethA-bearing mice. In the present study the effect of addition of human recombinant interleukin-2 (Hu-rIL-2) to the combination of Mu-rIFN beta with Mu-rIFN gamma on tumor growth in BALB/c mice bearing syngeneic MethA fibrosarcoma was examined. Low doses of Hu-rIL-2 (5 x 10(3) U or 5 x 10(4) U at 3-day intervals) showed no antitumor activity, while a high dose of Hu-rIL-2 (5 x 10(5) U) showed profound growth inhibition. The administration of IL-2 (ranging between 5 x 10(3) U and 5 x 10(5) U) in addition to the combination of IFN beta and IFN gamma showed more augmented antitumor effects in a dose-dependent manner. Furthermore, the simultaneous administration of IL-2, IFN beta and IFN gamma had more effective therapeutic activity, compared with the sequential administration of interferons and IL-2. These findings indicated that IL-2 in combination with IFN beta and gamma was effective for cancer treatment.

Animals↗
PubMed1990

A phase IA trial of sequential administration recombinant DNA-produced interferons: combination recombinant interferon gamma and recombinant interferon alfa in patients with metastatic renal cell carcinoma.

This study investigated the effects of sequentially administered recombinant interferon gamma (rIFN gamma) and recombinant interferon alfa (rIFN alpha) in 36 patients with metastatic renal cell carcinoma (RCC). rIFN alpha was subcutaneously administered daily for 70 days at dosages that varied (2.5, 5, 10, and 20 x 10(6) U/m2) across four cohorts of patients. Within each cohort of patients receiving a given dose of rIFN alpha, three subsets of patients received either 30, 300, or 1,000 micrograms/m2 rIFN gamma. rIFN gamma was administered intravenously for 5 days every third week, 6 hours prior to administration of rIFN alpha. Dose-limiting toxicity (DLT) included constitutional symptoms, leukopenia, nephrotic syndrome with acute renal failure, hypotension associated with death, and congestive heart failure. DLT was related more often to the rIFN alpha dose level than to rIFN gamma dose level. Maximum-tolerated dose (MTD) was 10 x 10(6) U/m2 rIFN alpha and 1,000 micrograms/m2 rIFN gamma. Six patients failed to complete a minimum of 21 days of therapy due to toxicity or rapid progression of disease. Clinical responses were seen in eight of 30 assessable patients. Two patients experienced complete remission and have remained in complete remission 20+ and 22+ months. An additional six patients have shown partial responses for 4 to 18+ months. One patient in partial remission continues to show slow regression of pulmonary and liver lesions off therapy with rIFNs. Clinical responses have remained durable for patients with complete remissions and patients with partial remissions. The results of this study suggest that toxicities associated with combination rIFN therapy can be reduced by administering these agents sequentially as opposed to simultaneously.

Adolescent↗
PubMed1992

RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur between replicating MHV RNAs and RNA fragments which do not replicate, suggesting the potential of RNA recombination for genetic engineering.

Base Sequence↗
PubMed1986

Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution.

We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor. Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII-BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region. We constructed a series of pMB080 deletions in vitro using exonucleases S1 and Bal31. Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction. Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system. The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids. We have determined the precise location of these recombination sites within oriV1 by DNA sequencing. These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base-pair inverted repeats found to be important for oriV1-oriV1 interactions. We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements. Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site-specific recombination process.

Base Sequence↗
PubMed1990

Human recombinant DNA-derived antihemophilic factor (factor VIII) in the treatment of hemophilia A. recombinant Factor VIII Study Group.

BACKGROUND: Current treatment of hemophilia A, a hereditary disorder affecting approximately 1 in 10,000 males, relies on plasma-derived factor VIII concentrates. We tested the safety and efficacy of a recombinant factor VIII preparation for the treatment of this disorder. METHODS: We conducted the investigation in three stages: comparing the pharmacokinetics of plasma-derived and recombinant factor VIII, assessing the efficacy of recombinant factor VIII for home therapy, and assessing its efficacy for major surgical procedures and hemorrhage. A total of 107 subjects with hemophilia, 20 of whom had not been treated previously, enrolled in the investigation. RESULTS: The in vivo recovery and elimination half-lives of recombinant factor VIII equaled or exceeded those of plasma-derived factor VIII. Seventy-six subjects participated in a home-treatment program, using recombinant factor VIII for 69 to 807 days (median, 618); home diaries of 56 subjects treated for 5 months were analyzed. Of 540 bleeding episodes, 399 (73.9 percent) required only one treatment with recombinant factor VIII. The projected annual consumption of recombinant factor VIII was similar to that of plasma-derived factor VIII concentrate. Twenty-six subjects received recombinant factor VIII for 22 surgical procedures and 10 serious hemorrhages; hemostasis was excellent in all cases. De novo formation of inhibitors occurred in only 1 of 85 previously treated subjects. Inhibitor antibodies also developed in 6 of 21 children, 20 of whom had not previously been treated; 5 had low levels (less than or equal to 7.5 Bethesda units) despite continued treatment with recombinant factor VIII. There was no evidence of new formation of antibody to foreign proteins, and recombinant factor VIII was well tolerated. CONCLUSIONS: Recombinant factor VIII has biologic activity comparable to that of plasma factor VIII and is safe and efficacious for the treatment of hemophilia A.

Adult↗
PubMed1979

Recombination nodules and synaptonemal complex in recombination-defective females of Drosophila melanogaster.

The cytological effects of mutant alleles of the mei-9, mei-218, and mei-41 loci during prophase I have been examined by electron microscopy. None of these mutants affect synaptonemal complex structure, continuity, or temporal behavior. Both the precondition-defective mutants mei-218 and mei-41 affect both number and morphology of spherical recombination nodules and apparently affect at least the numbers of ellipsoidal recombination nodules, whereas in the exchange-defective mutant mei-9 the numbers and morphologies of both ellipsoidal and spherical recombination nodules are normal. The parallel effects of mei-218 and mei-41 on meiotic recombination and on recombination nodules indicate that spherical recombination nodules at least mark the site of exchange events; the effects of these mutants on nodule morphology suggest that the nodule performs an active role in the recombination process. The nodule phenotype of mei-9 indicates that spherical nodules are present, and presumably functioning, well before the concluding stages of the recombination event. The parallel effects of all 3 mutants on ellipsoidal and spherical nodules indicate that these are indeed related structures but does not ellucidate the nature of the relationship. It is suggested that all aspects of meiotic recombination are under the aegis of recombination nodules.

Animals↗
PubMed1988

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.

Base Sequence↗
PubMed1992

Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays.

Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.

Base Sequence↗
PubMed1975

[Recombination of mitochondrial markers in yeasts. I. Analysis of isomitochondrial crosses and a study of the influence of locus determining mating type on recombination].

Yeast mitochondrial markers of the resistance to antibiotics (erythromycin, neomycin and monomycin) were used to study mitochondrial recombination in bifactorial crosses both in cis and trans configurations of markers. Our strains of Saccharomyces cerevisiae are isochromosomal and isomitochondrial due to all of them have originated from one haploid pace XII of Sacch. cerevisiae. The following parameters of mitochondrial recombination were examined in random samples of zygotic diploid daughter colonies; a) the asymmetric transmission of parental classes; b) the frequency of recombination; c) the polarity of recombination; d) the effect of cell mating type on these parameters in reciprocal crosses. The data obtained suggest the following features of mitochondrial recombination: a) The distribution of both classes as parental and recombinant is unequal: there is asymmetrical transmission (0,22-1,72) and some polarity of recombination (up to 4,0). b) The effect of cell mating type on asymmetrical transmission was established only is some crosses in trans-configuration of mitochondrial markers. In cis-configuration crosses such effect was not observed. c) Different mitochondrial mutations influenced on all the above listed parameters (asymmetrical transmission of parental classes, the frequency and the polarity of recombination). Some correlation between the high level of asymmetrical transmission of parental classes and the low frequency of recombination was found in crosses with NR and MR mutants. The existence of several different loci among ER, NR and MR mutants is suggested on the basis of the data on the recombination frequency.

Anti-Bacterial Agents↗
PubMed1986

The primary structure of crossover regions of intertypic poliovirus recombinants: a model of recombination between RNA genomes.

The nucleotide sequence of crossover sites in the genome of four intertypic (type 3/type 1) poliovirus recombinants has been determined. The approximate boundaries of the crossover regions were first estimated by RNase T1 oligonucleotide mapping of the recombinant genomes; then appropriate regions were sequenced by the chain termination method using oligonucleotide-primed reverse transcription of the recombinant RNAs. The crossover sites (defined as the contiguous sequences shared by the recombinant and both parental genomes flanked, in the recombinant genome, by heterotypic RNA segments) are 5, 5, 7, and 11 nucleotides long, respectively. The recombination was precise and was not accompanied by any other genetic alterations. The recombination sites were found to be located within genome segments having a potential to form secondary structure elements. Based on this observation, a model of recombination between picornaviral RNA genomes has been proposed. The essence of this model consists in bringing together homologous regions of two recombining RNA genomes via formation of intermolecular duplexes, detachment of the nascent 3' end of the newly synthesized complementary RNA from a "parting" site on the first template and its subsequent "jumping" to the identical (or closely related) "anchoring" site on the other template. Features of this model are discussed in some detail.

Base Sequence↗
PubMed1992

Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.

Animals↗
PubMed1977

[Recombination in amber mutants of bacteriophage T4B. III. Influence of X-mutation on intragenic recombination in T4 phages].

Study was made of the influence of X-mutation on interallelic and intergenic recombination at the region of genes 34-38 of phage T4B. In the case of intergenic crosses in all studied recombinations of genes mutation X caused a 2-fold decrease in recombination frequency. X-mutation showed the same effect on integenic recombination in genes 36 and 38. In the case of crossing flank markers of gene 35 the frequency of recombination decreased 3.5-fold, that of gene 34--6 fold. Crosses of double am-X mutants mapped at different sites of gene 34 allowed to detect the gradient of the effect X-mutation on recombination in gene 34 in the direction from gene 33 to gene 35. The data obtained indicate a local anomaly of DNA structure of the region of genes 34 and 35 and allowed to draw a conclusion that the degree of suppressing recombination process under the influence of X-mutation depends nor on the original recombination frequency between markers to be crossed, that is on the internal structure of the given region of the genome of bacteriophage T4. Possible reasons for specific effect of X-mutations on the recombination in genes 34 and 35 of phage T4B are discussed.

Alleles↗
PubMed1992

Response of CFU-GM (colony forming units for granulocytes and macrophages) from intact and pinealectomized rat bone marrow to murine recombinant interleukin-3 (rIl-3), recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) and human recombinant erythropoietin (rEPO).

The present study provides evidence, for the first time, of a role of the pineal gland in the proliferation of CFU-GM under the influence of different cytokines. The degree of colony formation in different cytokine groups was evaluated after 24 h, 3, 7 and 10 days. The colony growth in the present type of bone marrow cell cultures slows after the 10th day and ceases after the 14th day of incubation. The results show that rGM-CSF and the combination of rGM-CSF with rIl-3 and with rEPO stimulate the colony formation of granulocytes and macrophages. A 50% general reduction in the colony number was noted in the pinealectomy group. Their pattern of response to the different cytokines was similar to that of the intact group. It is suggested that the pineal activity has a physiological role in important aspects of host defence mechanisms, such as the proliferation of CFU-GM.

Animals↗