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PubMed1985

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.

Animals↗

PubMed2005

Effect of meiotic recombination on the production of aneuploid gametes in humans.

Within the last decade, aberrant meiotic recombination has been confirmed as a molecular risk factor for chromosome nondisjunction in humans. Recombination tethers homologous chromosomes, linking and guiding them through proper segregation at meiosis I. In model organisms, mutations that disturb the recombination pathway increase the frequency of chromosome malsegregation and alterations in both the amount and placement of meiotic recombination are associated with nondisjunction. This association has been established for humans as well. Significant alterations in recombination have been found for all meiosis I-derived trisomies studied to date and a subset of so called "meiosis II" trisomy. Often exchange levels are reduced in a subset of cases where the nondisjoining chromosome fails to undergo recombination. For other trisomies, the placement of meiotic recombination has been altered. It appears that recombination too near the centromere or too far from the centromere imparts an increased risk for nondisjunction. Recent evidence from trisomy 21 also suggests an association may exist between recombination and maternal age, the most widely identified risk factor for aneuploidy. Among cases of maternal meiosis I-derived trisomy 21, increasing maternal age is associated with a decreasing frequency of recombination in the susceptible pericentromeric and telomeric regions. It is likely that multiple risk factors lead to nondisjunction, some age dependent and others age independent, some that act globally and others that are chromosome specific. Future studies are expected to shed new light on the timing and placement of recombination, providing additional clues to the link between altered recombination and chromosome nondisjunction.

Aneuploidy↗

PubMed2000

Production and secretion patterns of cloned glucoamylase in plasmid-harboring and chromosome-integrated recombinant yeasts employing an SUC2 promoter.

To understand the differences in production and secretion patterns between plasmid-harboring and chromosome-integrated recombinant yeasts, the two recombinant Saccharomyces cerevisiae yeasts, containing the structural glucoamylase STA gene and the SUC2 promoter, were investigated. Both systems were regulated by glucose concentration in the culture broth. First, the glucoamylase activity per gene copy number of the chromosome-integrated recombinant yeast was 2.8- to 5.6-fold higher than that of the plasmid-harboring recombinant yeast. Overburdened owing to high copy number, the plasmid-harboring recombinant yeast gave lower glucoamylase activity per gene copy number. Second, the efficiency of signal sequence was compared; the secretion efficiency of glucoamylase in the plasmid-harboring recombinant yeast was higher than that in the chromosome-integrated recombinant yeast at 96 h of cultivation (74 vs 65%). We postulated that the higher level of secretion efficiency of the plasmid-harboring recombinant yeast resulted because the production level did not reach the capacity of the secretory apparatus of the host yeast. However, the specific secretion rate was much higher in the chromosome-integrated recombinant yeast even though the final secretion efficiency was lower. The lower secretion rate in the plasmid-harboring recombinant yeast could be explained by an adverse effect caused by higher production rate. Finally, the optimal glucose concentration for glucoamylase production in the chromosome-integrated recombinant yeast culture was lower than that in the plasmid-harboring recombinant yeast culture owing to gene dosage effect.

Cell Division↗

PubMed2004

Examination of the roles of Sgs1 and Srs2 helicases in the enforcement of recombination fidelity in Saccharomyces cerevisiae.

Mutation in SGS1, which encodes the yeast homolog of the human Bloom helicase, or in mismatch repair (MMR) genes confers defects in the suppression of mitotic recombination between similar but nonidentical (homeologous) sequences. Mutational analysis of SGS1 suggests that the helicase activity is required for the suppression of both homologous and homeologous recombination and that the C-terminal 200 amino acids may be required specifically for the suppression of homeologous recombination. To clarify the mechanism by which the Sgs1 helicase enforces the fidelity of recombination, we examined the phenotypes associated with SGS1 deletion in MMR-defective and recombination-defective backgrounds. Deletion of SGS1 caused no additional loss of recombination fidelity above that associated with MMR defects, indicating that the suppression of homeologous recombination by Sgs1 may be dependent on MMR. However, the phenotype of the sgs1 rad51 mutant suggests a MMR-independent role of Sgs1 in the suppression of RAD51-independent recombination. While homologous recombination levels increase in sgs1Delta and in srs2Delta strains, the suppression of homeologous recombination was not relaxed in the srs2 mutant. Thus, although both Sgs1 and Srs2 limit the overall level of mitotic recombination, there are distinct differences in the roles of these helicases with respect to enforcement of recombination fidelity.

DNA Helicases↗

PubMed2001

Recombinant human bone morphogenetic protein-2 accelerates healing in a rabbit ulnar osteotomy model.

BACKGROUND: Approximately 5% to 20% of fractures have delayed or impaired healing. Therefore, it is desirable to develop new therapies to enhance fracture-healing that can be used in conjunction with traditional treatment methods. The purpose of this study was to evaluate the ability of a single application of recombinant human bone morphogenetic protein-2 to accelerate fracture-healing in a rabbit ulnar osteotomy that heals spontaneously. METHODS: Bilateral mid-ulnar osteotomies (approximately 0.5 to 1.0 mm wide) were created in seventy-two skeletally mature male rabbits. The limbs were assigned to one of three groups: those treated with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2, those treated with an absorbable collagen sponge containing buffer, and those left untreated. In the first two groups, an 8 20-mm strip of absorbable collagen sponge containing either 40 g of recombinant human bone morphogenetic protein-2 or buffer only was wrapped around the osteotomy site. The rabbits were killed at two, three, four, or six weeks after surgery. In addition, twenty-four age-matched rabbits were used to provide data on the properties of intact limbs. The retention of recombinant human bone morphogenetic protein-2 at the osteotomy site was determined with scintigraphic imaging of (125)I-labeled recombinant human bone morphogenetic protein-2. After the rabbits were killed, the limbs were scanned with peripheral quantitative computed tomography to assess the area and mineral content of the mineralized callus. The limbs were then tested to failure in torsion, and undecalcified specimens were evaluated histologically. RESULTS: Gamma scintigraphy of (125)I-recombinant human bone morphogenetic protein-2 showed that 73% +/- 6% (mean and standard deviation) of the administered dose was initially retained at the fracture site. Approximately 37% +/- 10% of the initial dose remained at the site one week after surgery, and 8% +/- 7% remained after two weeks. The mineralized callus area was similar in all groups at two weeks, but it was 20% to 60% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 than in either the ulnae treated with buffer or the untreated ulnae at three, four, and six weeks (p < 0.05). Biomechanical properties were similar in all groups at two weeks, but they were at least 80% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 at three and four weeks than in either the ulnae treated with buffer (p < 0.005) or the untreated ulnae (p < 0.01). By four weeks, the biomechanical properties of the ulnae treated with recombinant human bone morphogenetic protein-2 were equivalent to those of the intact ulnae, whereas the biomechanical properties of both the ulnae treated with buffer and the untreated ulnae had reached only approximately 45% of those of the intact ulnae. At six weeks, the biomechanical properties were similar in all groups and were equivalent to those of the intact ulnae. The callus geometry and biomechanical properties of the ulnae treated with buffer were equivalent to those of the untreated ulnae at all time-points. CONCLUSIONS AND CLINICAL RELEVANCE: These findings indicate that treatment with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2 enhances healing of a long-bone osteotomy that heals spontaneously. Specifically, osteotomies treated with recombinant human bone morphogenetic protein-2 healed 33% faster than osteotomies left untreated. The results of this study provide a rationale for testing the ability of recombinant human bone morphogenetic protein-2 to accelerate healing in patients with fractures requiring open surgical management.

Animals↗

PubMed1995

B-complex recombinants and sarcoma regression: role of B-L/B-F region genes.

The anti-sarcoma response of three B complex recombinant haplotypes BR1(F24-G23), BR2(F2-G23), and BR3(F2-G23) was investigated. In a preliminary experiment, one male heterozygous for the BR1 recombinant haplotype and another heterozygous for the BR2 recombinant haplotype were each mated to females, some of which carried the respective recombinant. The anti-sarcoma response of progeny carrying the BR1 recombinant differed significantly from that of progeny carrying the BR2 recombinant. Subsequently, each of the three recombinant haplotypes was placed on each of four B haplotype complex backgrounds, and compared to B-G and B-L/B-F region controls on the same background haplotype. For each recombinant, significant differences in tumor growth were found between the recombinant and B-L/B-F control chickens on either one, two, or three of the four genetic backgrounds tested. For each recombinant, no differences were found between chickens carrying the recombinant and B-G region controls, which is further evidence that the gene(s) controlling Rous sarcoma growth lies in or near the B-L/B-F chromosomal region. Moreover, although the BR2 and BR3 recombinants appear to be identical serologically, they differed significantly in tumor growth suggesting that the two haplotypes are genetically different.

Animals↗

PubMed2002

Construction, expression and biological assessment of BPI23-Fcgamma1 recombinant protein prokaryotic expression vector.

OBJECTIVE: To construct pBV-BPI600-Fcgamma1(700) recombinant expression vector, to transform it into Escherichia coli DH5alpha, and to induce the expression of BPI23-Fcgamma1 anti-bacterial recombinant protein. METHODS: Genes coding for BPI23 and Fcgamma1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcgamma1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5alpha and BPI23-Fcgamma1 recombinant protein was expressed by a temperature-induced method. RESULTS: (1) Expected amplified products BPI600hp and Fcgamma1(700bp) were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcgamma1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. 3) pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcgamma1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcgamma1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization. CONCLUSION: pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and BPI23-Fcgamma1 recombinant protein with double biological activity of BPI and IgGFc was expresed in Escherichia coli.

Antimicrobial Cationic Peptides↗

PubMed1986

Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing.

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of plasmid-encoded FLP protein and two recombination sites on the plasmid. The recombination site possesses a specific orientation, which is determined by an asymmetric 8-base pair spacer sequence separating two 13-base pair inverted repeats. The outcome or directionality of site-specific recombination is defined by the alignment of two sites in the same orientation during the reaction. Sites containing point mutations or 1-base pair insertions or deletions within the spacer generally undergo recombination with unaltered sites at reduced levels. In contrast, recombination between the two identical mutant sites (where homology is restored) proceeds efficiently in all cases. Sites containing spacer sequences of 10 base pairs or more are nonfunctional under all conditions. A recombination site in which 5 base pairs are changed to yield an entirely symmetrical spacer sequence again recombines efficiently, but only with an identical site. This reaction, in addition, produces a variety of new products which can only result from random alignment of the two sites undergoing recombination, i.e. the reaction no longer exhibits directionality. These and other results demonstrate that both the efficiency and directionality of site-specific recombination is dependent upon homology between spacer sequences of the two recombining sites. This further implies that critical DNA-DNA interactions between the spacer region of the two sites involved in the reaction occur at some stage during site-specific recombination in this system. The specific spacer sequence itself appears to be unimportant as long as homology is maintained; thus, these sequences are probably not involved in recognition by FLP protein.

DNA↗

PubMed1993

Evidence for human meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19.

An improved linkage map for human chromosome 19 containing 35 short tandem repeat polymorphisms (STRPs) and one VNTR (D19S20) was constructed. The map included 12 new (GATA)n tetranucleotide STRPs. Although total lengths of the male (114 cM) and female (128 cM) maps were similar, at both ends of the chromosome male recombination exceeded female recombination, while in the interior portion of the map female recombination was in excess. Cosmid clones containing the STRP sequences were identified and were positioned along the chromosome by fluorescent in situ hybridization. Four rounds of careful checking and removal of genotyping errors allowed biologically relevant conclusions to be made concerning the numbers and distributions of recombination events on chromosome 19. The average numbers of recombinations per chromosome matched closely the lengths of the genetic maps computed by using the program CRIMAP. Significant numbers of chromosomes with zero, one, two, or three recombinations were detected as products of both female and male meioses. On the basis of the total number of observed pairs of recombination events in which only a single informative marker was situated between the two recombinations, a maximal estimate for the rate of meiotic STRP "gene" conversion without recombination was calculated as 3 x 10(-4)/meiosis. For distances up to 30 cM between recombinations, many fewer chromosomes which had undergone exactly two recombinations were observed than were expected on the basis of the assumption of independent recombination locations. This strong new evidence for human meiotic interference will help to improve the accuracy of interpretation of clinical DNA test results involving polymorphisms flanking a genetic abnormality.

Base Sequence↗

PubMed1993

Nondisjunction of chromosome 15: origin and recombination.

Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N = 27) and Angelman syndrome patients (N = 5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination are utilized. Standard methods of centromere mapping are employed to determine the level of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, most paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome.

Aneuploidy↗

PubMed1997

Characterization of recombination in the HLA class II region.

Studies of linkage disequilibrium across the HLA class II region have been useful in predicting where recombination is most likely to occur. The strong associations between genes within the 85-kb region from DQB1 to DRB1 are consistent with low frequency of recombination in this segment of DNA. Conversely, a lack of association between alleles of TAP1 and TAP2 (approximately 15 kb) has been observed, suggesting that recombination occurs here with relatively high frequency. Much of the HLA class II region has now been sequenced, providing the tools to undertake detailed analysis of recombination. Twenty-seven families containing one or two recombinant chromosomes within the 500-kb interval between the DPB1 and DRB1 genes were used to determine patterns of recombination across this region. SSCP analysis and microsatellite typing yielded identification of 127 novel polymorphic markers distributed throughout the class II region, allowing refinement of the site of crossover in 30 class II recombinant chromosomes. The three regions where recombination was observed most frequently are as follows: the 45-kb interval between HLA-DNA and RING3 (11 cases), the 50-kb interval between DQB3 and DQB1 (6 cases), and an 8.8-kb segment of the TAP2 gene (3 cases). Six of the 10 remaining recombinants await further characterization, pending identification of additional informative markers, while four recombinants were localized to other intervals (outliers). Analysis of association between markers flanking HLA-DNA to RING3 (45 kb), as well as TAP1 to TAP2 (15 kb), by use of independent CEPH haplotypes indicated little or no linkage disequilibrium, supporting the familial recombination data. A notable sequence motif located within a region associated with increased rates of recombination consisted of a (TGGA)12 tandem repeat within the TAP2 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

The recombinant limb as a model for the study of limb patterning, and its application to muscle development.

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning.

Animals↗

PubMed2004

Molecular control of recombination dynamics in dye-sensitized nanocrystalline TiO2 films: free energy vs distance dependence.

In this paper we address the dependence of the charge recombination dynamics in dye-sensitized, nanocrystalline TiO2 films upon the properties of the sensitizer dye employed. In particular we focus upon dependence of the charge recombination kinetics upon the dye oxidation potential E0(D+/D), determined electrochemically, and the spatial separation r of the dye cation HOMO orbital from the metal oxide surface, determined by semiempirical calculations. Our studies employed a series of ruthenium bipyridyl dyes in addition to porphyrin and phthalocyanine dyes. A strong correlation is observed between the recombination dynamics and the spatial separation r, with variation in r by 3 A resulting in a more than 10-fold change in the recombination half-time t(50%). This correlation is found to be in agreement with electron tunneling theory, t(50%) proportional, variant exp(-betar) with beta = 0.95 +/- 0.2 A-1. In contrast, the recombination dynamics were found to be relatively insensitive to variations in E0D+/D), indicative of the recombination reaction lying near the peak of the Marcus free energy curve, DeltaG approximately lambda, and with lambda approximately 0.8 eV. A correlation is also observed between the recombination half-time and the temporal shape of the kinetics, with faster recombination dynamics being more dispersive (less monoexponential). Comparison with numerical Monte Carlo type simulations suggests this correlation is attributed to a shift from fast recombination dynamics primarily limited by dispersive electron transport within the metal oxide film to slower dynamics primarily limited by the interfacial electron-transfer reaction. We conclude that the primary factor controlling the charge recombination dynamics in dye-sensitized, nanocrystalline TiO2 films is the spatial separation of the dye cation from the electrode surface. In particular, we show that for the Ru(dcbpy)2NCS2 dye series, the use of X = NCS rather than X = CN results in a 2 A shift in the dye cation HOMO orbital away from the electrode surface, causing a 7-fold retardation of the recombination dynamics, resulting in the remarkably slow recombination dynamics observed for this sensitizer dye.

Journal Article↗

PubMed1977

Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination.

Coliphages↗

PubMed1999

cDNA of the yeast retrotransposon Ty5 preferentially recombines with substrates in silent chromatin.

The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin. Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate. In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding. cDNA recombination requires Rad52p, which is responsible for homologous recombination. Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus. This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination. Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay. Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites. Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA. We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate. Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.

Amino Acid Sequence↗

PubMed1998

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis.

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Sinceper os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Animals↗

PubMed1999

Sensitive analysis of recombination activity using integrated cell surface reporter substrates.

BACKGROUND: Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result in various malignancies. So far the study of recombination activity has frequently relied on the use of reporter substrates that are limited by low sensitivity as well as tedious and distorting readout procedures. METHODS: Immunoglobulin class switch recombination substrates were generated which, upon recombination, resulted in the surface expression of human CD4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation. RESULTS: Recombining cells harboring integrated reporter substrates were analyzed by immunofluorescence and flow cytometry and could easily be isolated by high-gradient magnetic cell sorting (MACS). The analysis was not influenced by cloning efficiencies, as would be the case after drug selection, or prokaryotic recombination that might occur after analysis of recovered substrates in bacteria. In addition, cytometric readout is much faster, as it can be performed immediately after recombination. The substrate exhibited properties compatible with the detection of immunoglobulin class switch recombination and permitted the detection of recombination events down to 10(-5) per cell and generation. CONCLUSIONS: The high sensitivity of this system allows precise detection of very rare recombination events and thus permits the study of cell types with extremely low recombination activities.

Animals↗

PubMed1996

Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties.

Recombinant human liver phenylalanine hydroxylase (PAH) expressed in Escherichia coli has been purified to homogeneity. The recombinant enzyme exists in solution as a mixture of 80% tetramers and 20% dimers. A study of the kinetic properties of the enzyme indicates that compared to the recombinant and the native rat liver enzymes, the recombinant human enzyme is in an activated state. This conclusion is supported by the finding that its catalytic activity is only marginally stimulated by incubation with either phenylalanine or lysolecithin. In contrast, the native and the recombinant rat liver enzymes are activated 8- to 25-fold, respectively, when preincubated with phenylalanine or lysolecithin. In the absence of activators, the ratio of the hydroxylase activity in the presence of 6-methyl-5,6,7,8-tetrahydropterin compared to the activity in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), which is an index of the state of activation of the enzyme, is 4 for the human recombinant PAH compared to a value of 12 for the recombinant rat liver enzyme. Furthermore, the Km for phenylalanine in the presence of BH4 is 0.050 mM, a value that is one-fifth that of the recombinant rat liver enzyme. Covalent modification of the human enzyme by phosphorylation with protein kinase A provides further evidence that the human enzyme is in a substantially activated state. Phosphorylation, which results in the incorporation of 0.6 mol of phosphate/mol of subunit, leads to only a modest activation of 1.5-fold compared to about a 3-fold activation seen after phosphorylation of the native and the recombinant rat liver enzymes. Moreover, the recombinant human liver enzyme is less sensitive than the rat liver enzyme to stimulation by lysolecithin when tryptophan is the substrate. Just as is true for the rat liver enzyme, the apparent Km values for tryptophan and pheylalanine vary with the pterin cofactor employed. The ability of 7-tetrahydrobiopterin (7-BH4) to substitute for the natural cofactor tetrahydrobiopterin has been studied in vitro. The apparent Km for 7-BH4 for the recombinant human enzyme is 0.2 mM and the Km for phenylalanine is 0.05 mM. The hydroxylase reaction is severely inhibited by 7-BH4 in the presence of physiological concentrations of BH4. This inhibition can be overcome by a decrease in the concentration of phenylalanine. The implications of these novel properties of human PAH for phenylalanine homoestasis in man are discussed.

Animals↗