Physical and Genetic Characterization of a 75-Kilobase Deletion Associated with A(l), a Recessive Lethal Allele at the Mouse Agouti Locus

The agouti locus (A) of the mouse determines the timing and type of pigment deposition in the growing hair bulb, and several alleles at this locus are lethal when homozygous. Apparent instances of intragenic recombination and complementation between different recessive lethal alleles have suggested that the locus has a complex structure. We have begun to investigate the molecular basis of agouti gene action and recessive lethality by using a series of genetically linked DNA probes and pulsed field gel electrophoresis to detect structural alterations in radiation-induced agouti mutations. Hybridization probes from the Src and Emv-15 loci do not reveal molecular alterations in DNA corresponding to the ae, ax, and al alleles, but a probe from the parotid secretory protein gene (Psp) detects a 75-kilobase (kb) deletion in DNA containing the non-agouti lethal allele (al). The deletion is defined by a 75-kb reduction in the size of BssHII, NotI, NruI and SacII high molecular weight restriction fragments detected with the Psp probe and is located between 25 kb and 575 kb from Psp coding sequences. Because the genetic distance between A and Emv-15 is much less than A and Psp, there may be a preferred site of recombination close to Psp, or suppression of recombination between A and Emv-15. The al deletion has allowed us to determine the genotype of mice heterozygous for different recessive lethal alleles. We find that three different recessive lethal complementation groups are present at the agouti locus, two of which are contained within the al deletion.     

De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia

The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system.     

A Single Amino Acid Deletion in the Matrix Protein of Porcine Reproductive and Respiratory Syndrome Virus Confers Resistance to a Polyclonal Swine Antibody with Broadly Neutralizing Activity

Assessment of virus neutralization (VN) activity in 176 pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) identified one pig with broadly neutralizing activity. A Tyr-10 deletion in the matrix protein provided escape from broad neutralization without affecting homologous neutralizing activity. The role of the Tyr-10 deletion was confirmed through an infectious clone with a Tyr-10 deletion. The results demonstrate differences in the properties and specificities of VN responses elicited during PRRSV infection.     

A Genetic and Mosaic Analysis of a Locus Involved in the Anesthesia Response of Drosophila Melanogaster

We describe a genetic and behavioral analysis of several alleles of har38, a mutant with altered sensitivity to the general anesthetic halothane. We obtained a P-element-induced allele of har38 and generated several excision alleles by remobilizing the P element. The mutants narrow abdomen (na) and har85 are confirmed to be allelic to har38. Besides a decreased sensitivity to halothane, all mutant alleles of this locus cause a characteristic walking behavior in the absence of anesthetics. We have quantified this behavior using a geotaxis apparatus. Responses of the mutant alleles to different inhalational anesthetics were tested. The results strongly favor a multipathway model for the onset of anesthesia. Mosaic flies were tested for their response to halothane and checked for their abnormal walking behavior. The analysis suggests that both the behaviors are exhibited only by such mosaics as have the entire head of mutant origin. It is likely that this focus represents an element of a common pathway in the anesthetic response to several inhalational anesthetics but not all. This result is the first demonstration of regional specificity in the CNS of any animal for general anesthetic action.     

Deletion of the Murine Cytochrome P450 Cyp2j Locus by Fused BAC-Mediated Recombination Identifies a Role for Cyp2j in the Pulmonary Vascular Response to Hypoxia

Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human CYP2J2, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that Cyp2j gene products regulate the pulmonary vascular response to hypoxia.     

A Genetically Engineered Waterfowl Influenza Virus with a Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens

A deletion of about 20 amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza virus isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk deletion variant (MZ-delNA) were produced. Compared to the wild type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week-old chickens inoculated intratracheally with the MZ-delNA virus, viral replication in the lungs, liver, and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding interleukin-6 (IL-6), transforming growth factor-β4 (TGF-β4), and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver than did their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The measured enzymatic activities toward a small substrate were similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens.In the waterfowl reservoir, influenza A viruses are enzootic and infections are usually asymptomatic. The viruses replicate preferentially in the intestinal tract and are transmitted by the fecal-oral route. Phylogenetic analyses of amino acid changes show that influenza viruses in wild aquatic birds have low evolution rates, suggesting that they are in evolutionary stasis. Upon transmission to domestic poultry, rapid evolution occurs (63, 79). The replication of influenza viruses of duck origin in chickens is generally limited to the respiratory and gastrointestinal tracts and causes mild or no symptoms. However, sustained replication of viruses of the H5 or H7 subtype may lead to the emergence of highly pathogenic influenza viruses, which cause devastating epizootics (for a review, see reference 5). Mutations acquired upon replication in domestic poultry might also increase the potential for adaptation of avian influenza viruses to other species, including humans (20, 21, 50), which raises public health concerns.The viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are major determinants in the interspecies transmission and adaptation of influenza A viruses to a new host (for a review, see reference 46). The HA binds to the sialic acids (SA) linked to cellular membrane glycoproteins or glycolipids, whereas the sialidase activity of the NA facilitates the release and diffusion of progeny virions. SA are usually attached to a galactose moiety via an α2,3 or α2,6 glycosidic linkage. Avian viruses bind preferentially to SA-α2,3-galactose, whereas human viruses bind preferentially to SA-α2,6-galactose (13, 37, 56). This receptor binding specificity correlates with the relative predominance of SA-α2,3-galactose and SA-α2,6-galactose at the sites of viral multiplication in ducks and in humans, respectively (14, 28), and involves specific residues in the receptor binding site of the HA (37, 70). Viruses isolated from terrestrial poultry bind preferentially to SA-α2,3-galactose, but they differ from duck viruses by an enhanced binding to receptors in which the penultimate saccharide is sulfated and/or fucosylated (22, 23). Changes in the receptor binding site or at potential glycosylation sites of the HA that could modulate the binding of the HA to the receptors have been associated with the adaptation of duck viruses to poultry (3, 21, 36). A deletion of about 20 amino acids in the stalk region of the NA has also been frequently detected concomitant with the adaptation of duck viruses to poultry (3, 4, 25, 36, 62). In particular, it is a feature of the highly pathogenic H5N1 viruses that have become endemic in poultry in southern Asia since 2003 and have been causing sporadic human cases (26). The stalk is a flexible region which separates the globular, enzymatically active head of the tetrameric NA from the hydrophobic transmembrane domain (Fig. ​(Fig.1A).1A). The biological significance of the selection of variants with a shorter NA stalk is still unclear. Shortening of the stalk was found to decrease the ability of the NA to release the virus from cells (2, 6, 17, 25, 36, 39), and it was suggested that such a decreased activity of the NA could counterbalance a reduced binding of the HA to sialic acids expressed in poultry (2, 40, 71). The NA stalk length was found to have little effect on the efficiency of replication of a highly pathogenic H5N1 virus in poultry (39). However, it is possible that the presence of a multibasic site on the H5, a strong determinant of virulence, might be masking the effect, if any, of a shortened NA stalk.Open in a separate windowFIG. 1.Wild-type and deleted variants of the MZ-NA protein. (A) Schematic representation of the wild-type MZ-NA protein (470 amino acids [aa]). The domain corresponding to the stalk (amino acids 36 to 90) is represented in gray. The region of the stalk which is deleted in the MZ-delNA variant (amino acids 54 to 72) is represented by a hatched box. TM, transmembrane domain. (B) Amino acid sequence alignment of the NAs from MZ (H1N1), MZ-delNA (H1N1), A/Goose/Guandong/1/96 (H5N1), and A/Hong Kong/156/97 (H5N1) viruses. The domain corresponding to the stalk is highlighted in gray. The residues that are deleted in the NAs of MZ-delNA and A/Hong Kong/156/97 are represented by dashes.In the present study, we used a low-pathogenic duck influenza A(H1N1) virus isolate to investigate possible mechanisms involved in the selection of influenza viruses with a deletion in the stalk of the NA upon transmission from the waterfowl reservoir to chickens. Reverse genetics was used to produce a wild-type (wt) virus and a variant with a 19-amino-acid deletion in the stalk of the NA. The rationale for using an H1N1 virus was that a slight increase in viral fitness of the deleted variant, if any, would be more readily detected if the wild-type virus showed a low replication potential in chickens. In vitro and in vivo approaches were developed to examine the effect of the deletion in the stalk of the NA on the biological properties of the enzyme, on viral infectivity in avian cultured cells, and on viral infectivity and pathogenicity in chickens.     

Yip1A,a Novel Host Factor for the Activation of the IRE1 Pathway of the Unfolded Protein Response during Brucella Infection

Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments. 　On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation.     

Deletion of the Complement C5a Receptor Alleviates the Severity of Acute Pneumococcal Otitis Media following Influenza A Virus Infection in Mice

There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa^−/−) or factor B (Bf ^−/−) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection.     

Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production

The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant ΔabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the ΔabrA1/A2 strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.     

A rat homeobox gene, rNKx-2.5, is a homologue of the tinman gene in Drosophila and is mainly expressed during heart development

 We report the cloning of a rat homeobox-containing gene, rNkx-2.5, and investigation of its expression in adult tissues and during embryonic development. The rNkx-2.5 gene is a homologue of the tinman gene in Drosophila. Both genes share an identical TN domain (tinman-like amino-terminal decapeptide) and about 66.7% sequence identity within their homeodomain sequences. In vertebrates, the rNKx-2.5 gene is most closely related to the mouse NKx-2.5 (mNKx-2.5) gene. Coding sequences for both NKx-2.5 genes have 94.1% sequence identity, including three identical conserved domains, the TN, homeo and NK-2 domains (NK-2 specific domain, carboxy-terminal to the homeodomain in vertebrate tinman homologues). The rat NKx-2.5 gene is encoded by a 1.4-kb mRNA and in adult tissues is mainly expressed in heart, with weaker expression in testis, spleen and lung. During embryonic development, expression of rNKx-2.5 is strongly observed in developing heart, specifically in the myocardium of both atrial and ventricular chambers. In addition, rNKx-2.5 expression marks other developing tissues, including tongue, thyroid, stomach, spleen and pulmonary veins. These data show that rNKx-2.5 is expressed in a pattern similar but not identical to that previously observed for mNKx-2.5. Received: 24 February 1997 / Accepted: 23 June 1997     

Strategies for the Detection of Potential Beet necrotic yellow vein virus Genome Recombinations which might Arise as a Result of Growing A type Coat Protein Gene-expressing Sugarbeets in Soil Containing B Type Virus

We have searched for beet necrotic yellow vein virus (BNYVV) populations with a recombined genome which could possibly arise when transgenic sugarbeets expressing the coat protein gene of A type BNYVV are grown in soil containing Polymyxa betae carrying B type BNYVV, in soil samples from previous field release experiments and in a greenhouse model experiment. In order to accelerate the potential evolution of virus populations with recombined genomes in the model experiment, eight successive crops of sugarbeet plantlets were grown in the same soil samples over a period of 3 years. For the sensitive detection of recombined BNYVV genomes, we used nested PCRs with sense primers that are preferentially extended on the A type BNYVV sequence in the region of the coat protein gene and antisense primers which are preferentially extended on the B type BNYVV sequence in a region downstream of the coat protein gene which is not present in the transgene. Controls with mixtures of sap from plants which were singly infected with A or with B type BNYVV only revealed that, unless proper precautions are taken, PCR-mediated recombination artifacts may readily be produced. A method was developed that is able to detect A type/B type recombinant RNA molecules up to dilutions of one to a million in pure B type RNA molecules. Inspite of this high sensitivity we failed to detect any BNYVV with a recombined genome in the transgenic plants of the model experiment or at the sites of the previous field release experiments.