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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.
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Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.
The Hin site-specific recombination system normally promotes inversion of DNA between two recombination sites in inverted orientation. We show that the rate of deletion of DNA between two directly repeated recombination sites is 10-300 times slower than inversion between sites in their native configuration as measured in vivo and in vitro, respectively. In vitro studies have shown that the deletion reaction has the same requirement for Fis, a recombinational enhancer, and DNA supercoiling as the inversion reaction. These requirements, together with the finding that the deletion products are interlinked once suggest that the deletion synaptic complex is similar to the invertasome intermediate that generates inversion. The inefficiency of the deletion reaction is not a function of a reduced ability to recognize or synapse recombination sites in direct orientation. Not only do these substrates support an efficient knotting reaction, but directly repeated recombination sites with symmetric core sequences also invert efficiently. These findings demonstrate that the recombination sites are preferentially assembled into the invertasome structure with the sites aligned in the configuration for inversion regardless of their starting orientation. We propose that the dynamics of a supercoiled DNA molecule biases the geometric assembly of specific intermediates. In the case of Hin-mediated recombination, inversion is overwhelmingly preferred over deletion because DNA supercoiling favors a specific alignment of DNA strands in the synaptic complex.
The ability of recombinant interferons (IFNs) to modulate recombinant interleukin-2 (rIL-2) augmentation of natural killer (NK)-cell activity and to modulate the generation of activated killer (AK) cells was examined. Incubation of murine spleen cells for 18 hr with either human rIL-2 or a human hybrid recombinant IFN alpha, rHuIFN-alpha A/D, which is active on murine cells, resulted in a dose-dependent increase in NK activity; however, recombinant murine IFN gamma, rMuIFN-gamma, had little activity. A more than additive augmentation of cytotoxicity was obtained when spleen cells were incubated with the combination of rIL-2 and rHuIFN-alpha A/D. Incubation of murine spleen cells with rIL-2 for 3 days resulted in a dose-dependent induction of AK cells which were cytotoxic to an NK-resistant tumor target cell. In contrast to the results observed on NK activity, incubation of spleen cells with rHuIFN-alpha A/D and rIL-2 inhibited AK-cell activity. Partially purified murine IFN-alpha had inhibitory activity comparable to that of rHuIFN-alpha A/D. The addition of rHuIFN-alpha A/D at the initiation of culture of spleen cells with rIL-2 (day 0) resulted in maximal inhibition of cytotoxicity; inhibition was reduced or absent if rHuIFN-alpha A/D was added on day 1 or 2 of culture. The proliferation of spleen cells incubated with rIL-2 was also inhibited by rHuIFN-alpha A/D. Addition of rMuIFN-gamma to spleen-cells and rIL-2 increased the cytolytic activity of AK cells and did not inhibit rIL-2-induced proliferation of spleen cells. Similar data were also obtained with human peripheral blood lymphocytes and recombinant cytokines. Incubation of human peripheral blood lymphocytes with rIL-2 and recombinant human IFN-alpha A (rHuIFN-alpha A) or recombinant human IFN-gamma (rHuIFN-gamma) resulted in a more than additive increase in NK activity. Human AK-cell cytotoxicity was inhibited by rHuIFN-alpha A but enhanced by rHuIFN-gamma. Thus recombinant IFNs have differential effects on rIL-2-induced cytotoxic cells, resulting in augmentation or inhibition of activity, which is dependent on both the type of IFN and the cytotoxic activity examined. These results may have important implications for the potential therapeutic use of combinations of these cytokines.
The Gin recombinase of phage Mu selectively mediates DNA inversion between two inversely oriented recombination sites (gix) and requires the assistance of three accessory factors: negative supercoiling, an enhancer sequence, and the protein Fis. Deletion and fusion reactions are proscribed. Recombination by Gin is selective because it occurs only through a particular synaptic complex tailored for inversion. A single amino acid change in Gin allows it to carry out deletion and fusion as well as inversion and to dispense with the requirement for the accessory factors. We investigated the recombination mechanism of a mutant Gin protein by analyzing the knotted products of processive recombination by electron microscopy and gel electrophoresis. We find that, in sharp contrast to wild-type Gin, mutant Gin recombines through a broad spectrum of synaptic complexes that differ topologically. We propose a model for the selectivity of wild-type Gin recombination that explains how the dependence on the accessory factors limits recombination to inversion. In addition, we show that processive recombination by wild-type Gin is not restricted by the number of base-pairs separating the gix sites from each other and from the enhancer. This result can be explained if strand exchange proceeds through alternative paths dictated by the energetics of DNA coiling.
Three aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function. Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutations
To identify viral genes involved in reactivation of herpes simplex virus from latency, intertypic HSV-1 strain McKrae/HSV-2 strain HG 52 recombinants were selected following cotransfection of intact McKrae DNA and XbaI or HpaI cleaved HG 52 DNA. Eleven separately obtained recombinants containing HG 52 inserts between 0.35-0.56 and/or 0.82-1.0 map units (mu) were isolated. It was noted that with HpaI digested HG 52 DNA, only recombinants containing type 2 inserts from HpaI d (0.35-0.57) and/or containing an intact type 2 [S] region were isolated. Similarly with XbaI cleaved HG 52 DNA only recombinants containing type 2 sequences from XbaI c (0-0.45) were isolated. In effect, the type 2 insert always contained one or both origins of replication (ORIL/ORIS). In reciprocal experiments isolation of two recombinants from cotransfection of HpaI cleaved McKrae DNA with intact HG 52 DNA confirmed this finding; one contained both copies of ORIS and the intervening short region sequences of McKrae, the other contained approximately 3 kb of McKrae in which ORIL is located. These results indicate that either (a) the presence of an origin of replication in a RE fragment amplifies the fragment thereby increasing its concentration and hence recombination potential with intact genomes; and/or (b) recombination and replication may be correlated. In either case isolation of recombinants containing ORIL and ORIS from the restricted DNA parent strongly suggests that both origins are functional in vitro.
Recombination-defective female meiotic mutants representing 7 loci in Drosophila melanogaster have been examined for effects on gonial recombination in males. These loci were chosen for study because they represent a broad range of the known types of defects in processes necessary for meiotic recombination and somatic chromosome stability. Alleles at 6 of the loci studied did not increase the frequency of gonial recombination in males, whereas a mutant at one locus was associated with an increase (about 10-fold) in gonial recombination. These results suggest that the defects in chromosomal metabolism caused by these recombination, and in some cases repair, defective mutants are distinct from those of the male-recombination promoting elements (Mr) recently isolated from many natural populations. Analysis of the spontaneous events detected in this study showed that a third to a half of the events detected are actually of mutational rather than recombinational origin.
Overlap recombination has been used as a means of generating intertypic recombinants with crossover sites located within a defined region of the adenovirus genome. Using terminal DNA fragments of adenovirus type 2 and type 5 that overlap within the vicinity of the hexon coding region (51.6-59.7 map units), two different crosses could be studied; in one the overlap entirely encompasses the hexon and there are homologous regions at either side of the overlap where recombination is expected, and in the other only one side of the overlap is capable of sustaining recombination. The overall distribution of crossover sites within the overlap has been determined by restriction endonuclease mapping, and analysed in terms of the extent of homology between Ad2 and Ad5 in this region as defined by the DNA sequences (R. Kinloch, N. Mackay, and V. Mautner (1984). J. Biol. Chem., 259, 6431-6436; G. Akusjärvi, P. Aleström, M. Pettersson, M. Lager, H. Jörnvall, and U. Pettersson (1984). Submitted). Crossovers are found only in regions of relatively high DNA homology, as previously shown for intertypic recombination between temperature-sensitive viruses (M. E. G. Boursnell and V. Mautner (1981). Virology 112, 198-209). The presence of a free DNA end within the heterologous zone is insufficient to overcome the barrier to recombination. In crosses where recombination is confined to a relatively small homologous zone (45.9-53.0 mu) there is no special distribution of crossovers within the interval; no "hot spot" is discernible at the free DNA end, suggesting that a free DNA end is not especially recombinogenic, nor at the junction between the homologous and heterologous zones, suggesting that branch migration up to the heterology does not always occur. A cross designed to furnish evidence for gene conversion gave rise to a "conventional" recombinant with a crossover located within a 21-nucleotide tract of homology.
Recombinants with a centrally located crossover point were selected from crosses between poliovirus type 1 strains and intertypic (type 3/type 1) recombinants. Two such recombinants were characterized in some detail. In one of them (v1/a1-6), the 5' half of the genome was derived from a virulent type 1 strain, while the 3' half came from an attenuated type 1 strain. The genome of the other recombinant (a1/v1-7) had the reverse organization, with the 5' and 3' halves being derived from the type 1 attenuated and virulent strains, respectively. As deduced from the RNase T1 oligonucleotide maps, the a1/v1-7 genome also had a relatively short centrally located insert of the poliovirus type 3 origin. Both recombinants exhibited ts phenotypes. The RNA phenotypes of the recombinants corresponded to that of the parent donating the 3' half of the genome, v1/a1-6 and a1/v1-7 expressing RNA- and RNA +/- characters, respectively. Despite being a ts RNA- virus, v1/a1-6 proved to be neurovirulent when injected intracerebrally into Cercopithecus aethiops monkeys, although it exhibited a somewhat diminished level of pathogenicity as compared to its virulent type 1 parent. Recombinant a1/v1-7 behaved as an attenuated strain. These data supported our previous conclusion drawn from the experiments with intertypic poliovirus recombinants that the attenuated phenotype of poliovirus depends largely on the structure of the 5' half of its genome, although mutations of the 3' half may alleviate the virulence of the virus to a degree.
A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.