New genes originated via multiple recombinational pathways in the beta-globin gene family of rodents

Species differences in the size or membership composition of multigene families can be attributed to lineage-specific additions of new genes via duplication, losses of genes via deletion or inactivation, and the creation of chimeric genes via domain shuffling or gene fusion. In principle, it should be possible to infer the recombinational pathways responsible for each of these different types of genomic change by conducting detailed comparative analyses of genomic sequence data. Here, we report an attempt to unravel the complex evolutionary history of the beta-globin gene family in a taxonomically diverse set of rodent species. The main objectives were: 1) to characterize the genomic structure of the beta-globin gene cluster of rodents; 2) to assign orthologous and paralogous relationships among duplicate copies of beta-like globin genes; and 3) to infer the specific recombinational pathways responsible for gene duplications, gene deletions, and the creation of chimeric fusion genes. Results of our comparative genomic analyses revealed that variation in gene family size among rodent species is mainly attributable to the differential gain and loss of later expressed beta-globin genes via unequal crossing-over. However, two distinct recombinational mechanisms were implicated in the creation of chimeric fusion genes. In muroid rodents, a chimeric gamma/epsilon fusion gene was created by unequal crossing-over between the embryonic epsilon- and gamma-globin genes. Interestingly, this gamma/epsilon fusion gene was generated in the same fashion as the "anti-Lepore" 5'-delta-(beta/delta)-beta-3' duplication mutant in humans (the reciprocal exchange product of the pathological hemoglobin Lepore deletion mutant). By contrast, in the house mouse, Mus musculus, a chimeric beta/delta fusion pseudogene was created by a beta-globin --> delta-globin gene conversion event. Although the gamma/epsilon and beta/delta fusion genes share a similar chimeric gene structure, they originated via completely different recombinational pathways.     

Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1.

The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).     

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.     

The antiviral efficacy of the murine alpha-1 interferon transgene against ocular herpes simplex virus type 1 requires the presence of CD4(+), alpha/beta T-cell receptor-positive T lymphocytes with the capacity to produce gamma interferon

Alpha/beta interferons (IFN-alpha/betas) are known to antagonize herpes simplex virus type 1 (HSV-1) infection by directly blocking viral replication and promoting additional innate and adaptive, antiviral immune responses. To further define the relationship between the adaptive immune response and IFN-alpha/beta, the protective effect induced following the topical application of plasmid DNA containing the murine IFN-alpha 1 transgene onto the corneas of wild-type and T-cell-deficient mice was evaluated. Mice homozygous for both the T-cell receptor (TCR) beta- and delta-targeted mutations expressing no alpha beta or gamma delta TCR (alpha beta/gamma delta TCR double knockout [dKO]) treated with the IFN-alpha 1 transgene succumbed to ocular HSV-1 infection at a rate similar to that of alpha beta/gamma delta TCR dKO mice treated with the plasmid vector DNA. Conversely, mice with targeted disruption of the TCR delta chain and expressing no gamma delta TCR(+) cells treated with the IFN-alpha 1 transgene survived the infection to a greater extent than the plasmid vector-treated counterpart and at a level similar to that of wild-type controls treated with the IFN-alpha 1 transgene. By comparison, mice with targeted disruption of the TCR beta chain and expressing no alpha beta TCR(+) cells (alpha beta TCR knockout [KO]) showed no difference upon treatment with the IFN-alpha1 transgene or the plasmid vector control, with 0% survival following HSV-1 infection. Adoptively transferring CD4(+) but not CD8(+) T cells from wild-type but not IFN-gamma-deficient mice reestablished the antiviral efficacy of the IFN-alpha 1 transgene in alpha beta TCR KO mice. Collectively, the results indicate that the protective effect mediated by topical application of a plasmid construct containing the murine IFN-alpha 1 transgene requires the presence of CD4(+) T cells capable of IFN-gamma synthesis.     

Expression of the Viral Thymidine Kinase Gene in Herpes Simplex Virus-Transformed L Cells

In these studies, the expression of thymidine kinase (TK) in normal and herpes simplex virus (HSV)-transformed L cells has been compared. In asynchronously dividing cultures of L cells, the TK activity rose and declined rapidly and coordinately with DNA synthesis. When net cell increase stopped, TK activity was at a minimum. In contrast, TK activity of HSV-transformed cells remained at a minimum during rapid DNA synthesis and gradually increased as the rate of DNA synthesis decreased. When net cell increase stopped, TK activity was at a maximum. In synchronous cultures of L cells, TK activity rose and fell coordinately with the rate of DNA synthesis. In synchronous cultures of HSV-transformed cells, no increase in TK activity was observed during the period of rapid DNA synthesis, i.e., the S phase. These findings indicated that the viral TK gene in HSV-transformed cells was not placed under the control of the cellular mechanisms which normally modulate the host cell TK gene. Lytic infection of HSV-transformed cells with a TK(-) mutant of HSV-1 induced a four-to fivefold increase in viral TK. The TK of HSV-1 was induced in the HSV-1-transformed cells and HSV-2 in the HSV-2-transformed cells by this TK(-) mutant. The same infection of normal L cells decreased the cellular TK activity by 80%. This stimulation, rather than inhibition, suggest that the viral gene in HSV-transformed cells retain some of its original viral characteristics.     

Mutational analysis of the ICP4 binding sites in the 5'' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene.

A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.     

Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice.

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.     

Expression of chimeric human beta- and delta-globin genes during erythroid differentiation

To determine whether sequences contained within the small intervening sequence (IVS 1) or large intervening sequence (IVS 2) are involved in the regulated expression of the human beta-globin gene, chimeric genes containing portions of the human beta- and delta-globin genes were stably transfected into mouse erythroleukemia (MEL) cells. Since MEL cells can be induced to differentiate in culture, the expression of the chimeric genes was compared to the expression of beta and delta both before and after the induction of erythroid differentiation. The expression of beta delta 1, a beta-globin gene containing delta IVS 1 in place of beta IVS 1, was comparable to the expression of a beta-globin gene both before and after erythroid differentiation. However, the base-line expression of human beta-globin genes containing delta IVS 2 in place of beta IVS 2 was dramatically decreased. Furthermore, the substitution of delta IVS 2 for beta IVS 2 prevented the regulated increase in expression of the beta-globin gene upon induction. The results also indicate that sequences present in beta IVS 2 are not sufficient for this induced increase in expression since the substitution of beta IVS 2 for delta IVS 2 in a delta gene does not increase the regulated expression of delta during differentiation. These experiments suggest that either the presence of delta IVS 2 in a beta gene interrupts sequences required for the induced expression of beta-globin or that sequences in beta IVS 2 act in concert with other beta globin sequences not present in the delta-globin gene to permit optimal expression.     

Regulation of glycoprotein D synthesis: does alpha 4, the major regulatory protein of herpes simplex virus 1, regulate late genes both positively and negatively?

Earlier studies have described the alpha 4/c113 baby hamster kidney cell line which constitutively expresses the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1). Introduction of the HSV-1 glycoprotein B (gB) gene, regulated as a gamma 1 gene, into these cells yielded a cell line which constitutively expressed both the alpha 4 and gamma 1 gB genes. The expression of the gB gene was dependent on the presence of functional alpha 4 protein. In this article we report that we introduced into the alpha 4/c113 and into the parental BHK cells, the HSV-1 BamHI J fragment, which encodes the domains of four genes, including those of glycoproteins D, G, and I (gD, gG, and gI), and most of the coding sequences of the glycoprotein E (gE) gene. In contrast to the earlier studies, we obtained significant constitutive expression of gD (also a gamma 1 gene) in a cell line (BJ) derived from parental BHK cells, but not in a cell line (alpha 4/BJ) which expresses functional alpha 4 protein. RNA homologous to the gD gene was present in significant amounts in the BJ cell line; smaller amounts of this RNA were detected in the alpha 4/BJ cell line. RNA homologous to gE, presumed to be polyadenylated from signals in the vector sequences, was present in the BJ cells but not in the alpha 4/BJ cells. The expression of the HSV-1 gD and gE genes was readily induced in the alpha 4/BJ cells by superinfection with HSV-2. The BJ cell line was, in contrast, resistant to expression of HSV-1 and HSV-2 genes. The BamHI J DNA fragment copy number was approximately 1 per BJ cell genome equivalent and 30 to 50 per alpha 4/BJ cell genome equivalent. We conclude that (i) the genes specifying gD and gB belong to different viral regulatory gene subsets, (ii) the gD gene is subject to both positive and negative regulation, (iii) both gD and gE mRNAs are subject to translational controls although they may be different, and (iv) the absence of expression of gD in the alpha 4/BJ cells reflects the expression of the alpha 4 protein in these cells.     

Molecular genetics of herpes simplex virus. VII. Characterization of a temperature-sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of gamma polypeptides.

We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 39 degrees C relative to that at 33.5 degrees C was 5 X 10(-6). The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that alpha and beta polypeptides were produced, whereas most gamma polypeptides were either reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43, and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 10(3) base pairs between 0.383 and 0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 molecular weight), which was previously identified as a beta polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells.     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

DNA sequences regulating human beta globin gene expression.

Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression.