Temperature regulates NF-κB dynamics and function through timing of A20 transcription.

NF-κB signaling plays a pivotal role in control of the inflammatory response. We investigated how the dynamics and function of NF-κB were affected by temperature within the mammalian physiological range (34 °C to 40 °C). An increase in temperature led to an increase in NF-κB nuclear/cytoplasmic oscillation frequency following Tumor Necrosis Factor alpha (TNFα) stimulation. Mathematical modeling suggested that this temperature sensitivity might be due to an A20-dependent mechanism, and A20 silencing removed the sensitivity to increased temperature. The timing of the early response of a key set of NF-κB target genes showed strong temperature dependence. The cytokine-induced expression of many (but not all) later genes was insensitive to temperature change (suggesting that they might be functionally temperature-compensated). Moreover, a set of temperature- and TNFα-regulated genes were implicated in NF-κB cross-talk with key cell-fate-controlling pathways. In conclusion, NF-κB dynamics and target gene expression are modulated by temperature and can accurately transmit multidimensional information to control inflammation.

Studies of genetic transmission of murine leukemia virus by AKR mice. I. Crosses with Fv-1 n strains of mice.

AKR mice, which regularly contain infectious murine leukemia virus, were mated with four Fv-1(n) strains of mice which show little or no expression of virus. F(1), F(2), and first and second backcross generation hybrids were tested for virus in tail tissue at 2 and 6 wk of age. The segregation data indicate that the AKR mouse contains two unlinked, autosomal, chromosomal loci, either of which suffices to induce detectable levels of infectious virus in Fv-1(n) progeny by 6 wk of age. One of the loci (tentatively referred to as V(1)) is on linkage group I, 25-30 map units from the locus for albino; the gene order tentatively appears to be N(1)-c-Hbb.

Quantitative studies of naturally occurring murine leukemia virus infection of AKR mice.

Quantitative studies were made of the organ distribution of murine leukemia virus in AKR mice of various ages. Infectious virus first appeared shortly before or after birth and was continuously present in all mice thereafter. Highest infectivity titers were found in uterus and bone, with spleen slightly lower. Virus titers in normal thymus were relatively low, but increased significantly with the development of thymic lymphoma. The level of viremia decreased after the 1st month of life, but increased sharply in lymphomatous mice.

Abelson virus-induced lymphomagenesis in mice.

The salient facts which have emerged from our study of Abelson virus (MuLV-A)lymphomagenesis in mice are that lymphoma induction is (a) age dependent, (b) virus dose dependent, and (c) under the control of host genes unrelated to other genes known to control murine leukemia (e.g., Fv-1 on Fv-2). Of 16 strains tested, only BALB/c and some of its derivative strains showed high sensitivity. Studies from CXB recombinant inbred strains and hybrids between them are interpreted to indicate that BALB/c carries dominant sensitivity alleles at two loci, tentatively designated Av-1 and Av-2, which conferon these mice partial susceptibility to MuLV-A lymphoma induction. In addition, H-2 may play a minor role in determining the susceptibility of mice to MuLV-A, its effect being seen only in mice homozygous for resistance at both Av-1 and Av-2. Virologic studies indicate that the resistance of adult B6 mice is not related to restriction on the helper virus replication, but is specific for the defective transforming virus genome.

Genes affecting mink cell focus-inducing (MCF) murine leukemia virus infection and spontaneous lymphoma in AKR F1 hybrids.

An assessment of the importance of mink cell focus-inducing (MCF)-type recombinant murine leukemia viruses in spontaneous thymic lymphomagenesis and of the genetic factors affecting its occurrence was carried out with F1 hybrids between AKR and various other inbred strains. There was generally close agreement between the frequency of detection of MCF virus, of thymocyte antigenic amplification in the preleukemic period, and of spontaneous lymphoma. Also, hybrid combinations with moderate to high spontaneous lymphoma were uniformly susceptible to lymphoma induction by neonatal inoculation of MCF 247 virus, while lower leukemic hybrids were at least partially resistant to the induced disease. At least four resistance genes can be identified as affecting the disease in the various hybrids: Fv-1, Rmcf, an unidentified gene carried by the C57 series of mice and SJL, and an unidentified minor gene carried by several other strains.

A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses.

Previous studies have indicated that all naturally occurring murine leukemia viruses propagate significantly more efficiently on embryo cells of either NIH Swiss or BALB/c mice. Studies of the plaquing efficiency of representative viruses on embryo cells of various inbred and hybrid mice indicate that the pattern of sensitivity of the cells is genetically determined. All of 23 strains tested were found to resemble either NIH Swiss (N-type) or BALB/c (B-type) with respect to plaquing efficiency of these viruses. Virus growth on embryo cells derived from (N-type x B-type)F(1) hybrids indicated dominance of resistance to both types of viruses. Backcross hybrid studies indicated that a single locus is the primary determinant of the host-range patterns observed. This locus has no effect on growth of certain laboratory-passaged leukemia viruses which propagate equally well on embryo cells of all mouse strains, F(1), and backcross hybrids. Though other genetic and nongenetic factors influence viral growth or expression in vitro and in vivo, the genetic locus described appears of major significance in the biology of murine leukemia.

A major genetic locus affecting resistance to infection with murine leukemia viruses. II. Apparent identity to a major locus described for resistance to friend murine leukemia virus.

The N-B locus affecting tissue culture infectivity with naturally occurring murine leukemia viruses appears to be identical to the Fv-1 locus described for sensitivity to Friend leukemia virus. Results of tissue culture studies were parallel to results of studies in vivo and indicate that the F-S virus is N-tropic and the F-B virus is NB-tropic. Inbred and partially congenic mouse strains sensitive at Fv-1 show N-type sensitivity; strains resistant at Fv-1 show B-type sensitivity. The Fv-2 locus does not appear to exert significant effect in tissue culture. Knowledge of N-B type has been useful in predicting Fv-1 sensitivity.

Genetic mapping of ecotropic murine leukemia virus-inducing loci in six inbred strains.

Mendelian segregation analysis was used to map chromosomal genes for the induction of endogenous N- and B-tropic ecotropic retroviruses (V loci) in high and low leukemic mouse strains. Patterns of virus expression were determined for mice of various inbred strains, congenic lines carrying single V loci, and the linkage testing stocks used in mapping studies. Segregation analysis resulted in the genetic mapping of V loci from six inbred strains to five mouse chromosomes. The V locus of A/J was mapped to chromosome 5 and shown to be allelic with that of BALB/cJ and C3H/HeJ (Cv); this suggests that Cv-represents a stable ancestral V locus present in Bagg albino stocks before the separation of inbred lines. The single, poorly inducible V locus of C57BL/10J and one of the four high virus loci of C58/Lw were mapped to the same region of chromosome 8 and may represent an allelic pair with different patterns of expression. An N-tropic V locus of the SEA/GnJ mouse was mapped to chromosome 9, and one of the three V loci of C3H/FgLw was mapped to chromosome 7. The endogenous B-tropic virus of B10.BR/SgLi was mapped to chromosome 11. These studies provide further evidence that endogenous ecotropic V loci are present at different chromosomal sites in unrelated mouse strains and emphasize the role of germ line reinfections in the generation of this diversity.

Genetic mapping of the ecotropic virus-inducing locus Akv-2 of the AKR mouse.

A combination of somatic cell hybridization and standard mendelian breeding techniques was used to map the AKR ecotropic virus inducibility locus Akv-2 to the centromeric end of chromosome 16. This assignment of Akv-2 further emphasizes the endogenous ecotropic retroviruses are inserted at multiple sites in mouse chromosomes.

Genetic mapping of xenotropic murine leukemia virus-inducing loci in five mouse strains.

A single mendelian gene was identified for induction of the endogenous xenotropic murine leukemia virus in five mouse strains (C57BL/10, C57L, C57BR, AKR, and BALB/c). This locus, designated Bxv-1, mapped to the same site on chromosome 1 in all strains: Id-1-Pep-3-[Bxv-1-Lp]. Thus, inducibility loci for xenotropic virus are more limited in number and chromosomal distribution than ecotropic inducibility loci. Virus expression in mice with Bxv-1 was induced by treatment of fibroblasts with 5-iododeoxyuridine or by exposure of spleen cells to a B cell mitogen, bacterial lipopolysaccharide. An analysis of the hamster X mouse somatic cell hybrids indicated that chromosome 1, alone, was sufficient for virus induction.

Studies of genetic transmission of murine leukemia virus by AKR mice. II. Crosses with Fv-1 b strains of mice.

The transmission of murine leukemia virus (MLV) to hybrids between AKR and Fv-1(b) mice was studied in order to evaluate the effect of the Fv-1 gene on endogenous MLV infection and to attempt to determine if the genetic loci contributed by AKR carry viral genetic determinants. Fv-1 was shown to have a marked suppressive effect on time of appearance of detectable infectious virus and on the titers attained in vivo, but did not affect the ability of the cells to produce virus in vitro after induction with 5-iododeoxyuridine. The host range type of the virus detected in the hybrid mice was almost always of the type carried by AKR, although the low-virus Fv-1(b) parents carry the genome of a different host range type. This finding provides strong, but not conclusive, evidence that the virus-inducing loci of AKR contain MLV genetic determinants.

A major genetic locus affecting resistance to infection with murine leukemia viruses. 3. Assignment of the Fv-1 locus to linkage group 8 of the mouse.

The Fv-1 gene, which regulates sensitivity of mouse cells to infection by naturally occurring host-range types of murine leukemia virus, was shown to be located on linkage group VIII (chromosome 4), 39 map units from b.

Cell-surface antigens associated with recombinant mink cell focus-inducing murine leukemia viruses.

Distinct type-specific antigens were detected on cells infected with cloned mink cell focus-inducing (MCF) murine leukemia viruses by means of cell surface immunofluorescence absorption assays with rabbit antisera raised against naturally-occurring AKR MCF viruses. The MCF type-specific antibodies were present in high titer and not absorbable by cells infected with ecotropic, xenotropic, or wild mouse amphotropic murine leukemia viruses, or combinations of ecotropic and xenotropic viruses. Three MCF subtype-specific reactions were identified. One subspecificity (operationally designated MCFA-1) defined antigenic determinant(s) distributed among MCF viruses in general. Another (MCFA-2) specified determinant(s) induced by all naturally occurring MCF isolates not of Friend or Moloney origin. A third subspecificity (MCFA-3) was induced by some MCF isolates, and not by others; the presence of this antigen did not correlate with the source of any presently known biological property of the viruses. In addition, type-specific antigenic determinants of ecotropic and xenotropic murine leukemia viruses were expressed on MCF virus-infected cells. The serological profile of MCF viruses thus supports the contention that they are env gene recombinants between ecotropic and xenotropic murine leukemia viruses. However, new, distinct MCF-specific determinants are also generated, and these could be useful markers in studying MCF viruses.

Genetic study of lymphoma induction by AKR mink cell focus-inducing virus in AKR x NFS crosses.

The mink cell focus-inducing (MCF)-247 virus, originally isolated from an AKR thymoma, is lymphomagenic in AKR mice but not in the ecotropic virus-negative NFS mouse strain. Analysis of sensitivity to lymphoma-induction by AKR-247 MCF virus in genetic hybrids between these two strains showed that F1 mice inoculated as sucklings were uniformly sensitive, whereas those inoculated as weanlings were generally resistant. In NFS backcross mice inoculated as sucklings, inheritance and expression of endogenous ecotropic virus from AKR was an essential correlate of replication of MCF virus and subsequent development of lymphoma. However, one-third of the mice expressing ecotropic virus and replicating the inoculated MCF virus did not develop lymphoma. The results suggested that an additional gene that influenced development of lymphoma may be involved, and that mice inheriting both virus-inducing loci from AKR were more susceptible than those inheriting only one. These findings indicate that the causal role of ecotropic virus infection in spontaneous thymomagenesis in AKR mice involves not only the generation of leukemogenic MCF viruses but also the establishment of permissiveness for their growth.

Correlation of early murine leukemia virus titer and H-2 type with spontaneous leukemia in mice of the BALB/c times AKR cross: a genetic analysis.

Tissue extracts from 6-wk old mice of the AKR strain (H-K) show high levels of infectious murine leukemia virus, and these mice show a near 100% incidence of spontaneous leukemia. In F1 mice of the cross, BALB/c times AKR (H-2K/H-2K), both the occurrence of virus and the incidence of spontaneous leukemia are suppressed to very low values, due largely to the presence of the FV-1b allele inherited from the BALB/c parent. Mice of the (BALB/c times AKR) F-1 times AKR backcross generation were observed for possible correlations between virus expressions at 6 wk of age, H-2 type and leukemia incidence. H-2 type showed at most a weak influence on the occurrence of infectious virus, but there was a very strong correlation between the level of virus expression and the occurrence of leukemia and a strong correlation between H-2 type and leukemia. In addition, there was a highly significant nonrandom distribution of virus-negative mice among the backcross litters, suggesting a maternal effect on virus expression.

Lymphomagenicity of recombinant mink cell focus-inducing murine leukemia viruses.

Recombinant mink cell focus-inducing (MCF) murine leukemic viruses, as well as ecotropic and xenotropic viruses, were tested for ability to accelerate or cause development of lymphoma in AKR and other strains of mice. Of the three classes of virus isolated from AKR, only the MCF viruses were able to accelerate development of AKR lymphoma. This fully supports the idea that the MCF viruses are the proximal cause of spontaneous AKR lymphoma. MCF lymphomagenicity was strain specific, however, in that AKR MCF viruses did not induce lymphomas in many murine strains; they were moderately lymphomagenic in C3H/Bi mice and in National Institutes of Health Swiss partially congenic for Akv-1 or Akv-2. In contrast, MCF viruses from nonthymic hematopoietic neoplasms of C3H/Fg, BALB/c, or mice partially congenic for ecotropic virus loci (Akv-1, Akv-2, Fgv-1, C58v-1, and C58v-2) were not able to accelerate or cause lymphomia in AKR or any other mouse strain tested, including some of the strains of origin. MCF lymphomagenicity correlated with thymic origin in the virus and with ability to replicate in the thymus.

Immunofluorescent studies of the potentiation of an adenovirus-associated virus by adenovirus 7.

A quantitative immunofluorescent procedure for detection of viral antigen was used to study the potentiation of AAV-1 by Ad.7. AAV viral antigen formed only when the cells were also infected with adenovirus, and only in cell culture systems in which the adenovirus infection proceeded to completion. Ad. 7 infection of AGMK. cell cultures did not potentiate AAV unless the Ad. 7 infection was itself potentiated by SV40. Dose-response studies indicated that a single AAV particle and a single infectious Ad. 7 particle sufficed to initiate AAV antigen synthesis. Sequential inoculation studies showed that AAV antigen formed simultaneously with Ad. 7 viral antigen when the AAV was inoculated any time between 15 hr before to 10 hr after the Ad. 7, both antigens appearing about 15 hr after inoculation of Ad. 7. The AAV-1 antigen formation had a minimum latent period of 5 hr, as seen with Ad. 7 preinfection of 10 hr or more. When UV-irradiated Ad. 7 was used as helper, the AAV antigen still appeared simultaneously with the Ad. 7 viral antigen, even though the latter was delayed by 23 hr compared to nonirradiated virus. When the early replicative events of both viruses were allowed to proceed in FUDR-inhibited cells, and then the FUDR inhibition was reversed, AAV antigen formed within 2 hr, which was 3 hr before the Ad. 7 viral antigen appeared. It was inferred that the event in the adenovirus cycle that renders a cell competent to synthesize AAV occurs after the 10th hr and may be temporally associated with replication of the adenovirus DNA.

Relationship of infectious murine leukemia virus and virus-related antigens in genetic crosses between AKR and the Fv-1 compatible strain C57L.

In a further genetic study of murine leukemia virus (MuLV) and its components we examined the backcross C57L X (C57L X AKR). This population was selected because strains AKR and C57L are both Fv-1n, and the restriction which the Fu-1b allele imposes on the output of virus was thereby obviated. The segregants were scored for three characters: (a) infectious Gross-AKR-type MuLV (V), in the tail; (b) group-specific antigen indicative of p30 internal viral protein, in spleen; and (c) GIX antigen, now thought to be indicative of gp69/71 viral envelope glycoprotein, on thymocytes. Our conclusions are: (a) It is confirmed that the AKR mouse has two unlinked chromosomal genes, Akv-1 and Akv-2, each of which can independently give rise to the life-long high output of MuLV that is characteristic of AKR mice. (b) Of the eight phenotypes that could possibly be derived from segregation of the three pairs of independent alternative traits, seven were observed, but on progeny testing only three were shown to reflect stably heritable genotypes; these were V+p30+GIX+ and V-p30-GIX- (the parental types) and V-p30+GIX+. A third, newly identified AKR gene, designated Akvp, segregating independently of Akv-1 and Akv-2, also determines expression of p30 and GIX but in this case independently of XC-detectable MuLV. (c) The four remaining observed phenotypes, which did not breed true on progeny testing, involved mostly antigen-negative parents yielding antigen-positive progeny; it is likely that these discrepancies represented suppression of phenotype by a maternal resistance factor.

Relation of chromosome 4 (linkage group 8) to murine leukemia virus-associated antigens of AKR mice.

Genes specifying or controlling the expression of G(IX) (cell surface), GCSA (cell surface), and gs (internal viral) antigens are located in chromosome 4 (linkage group [LG] VIII) of the AKR mouse. All three antigens may exhibit mendelian inheritance, mice being antigen positive or antigen negative, but each may also appear in leukemic cells of mice whose inherited genotype was antigen negative. The G(IX)-determining gene in LG VIII of AKR mice apparently is equivalent to Gv-1, which determines expression of the same antigen in 129 strain mice, but which in the latter strain is located in LG IX. As the estimated distance of Gv-1 from H-2 in 129 mice is considerable (37 units) further tests are now indicated to assess the possibility of pseudolinkage in this case. The Fv-1 locus, also located in LG VIII, influences the mouse's titer of MuLV, and might thereby be thought to regulate the G(IX) and gs phenotypes of AKR backcross segregants. But the data indicate a discrete LG VIII locus for G(IX), since expression of this antigen is mendelian and independent of infectious virus titer. Since the G(IX) and GCSA phenotypes of AKR backcross segregants were invariably concordant, these two antigens must be specified or controlled by closely linked genes, and the latter also is presumably independent of virus titer. The question as to what extent expression of gs antigen in the segregants is secondary to virus production is undecided.

Convergent evolution to an aptamer observed in small populations on DNA microarrays.

The development of aptamers on custom synthesized DNA microarrays, which has been demonstrated in recent publications, can facilitate detailed analyses of sequence and fitness relationships. Here we use the technique to observe the paths taken through sequence-fitness space by three different evolutionary regimes: asexual reproduction, recombination and model-based evolution. The different evolutionary runs are made on the same array chip in triplicate, each one starting from a small population initialized independently at random. When evolving to a common target protein, glucose-6-phosphate dehydrogenase (G6PD), these nine distinct evolutionary runs are observed to develop aptamers with high affinity and to converge on the same motif not present in any of the starting populations. Regime specific differences in the evolutions, such as speed of convergence, could also be observed.