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In the present paper, bovine serum albumin (BSA) was studied for extraction case from a buffer solution through a new version of modified magnetic chitosan nanocomposite. Post-modification of this magnetic chitosan led to conversion of amine groups to dithiocarbamate on the surface of chitosan which was wrapped to superparamagnetic iron oxide nanoparticles (SPION). Chitosan was converted to magnetic chitosan over co-precipitation of Fe2+ and Fe3+ under alkali conditions. Amines of chitosan were also converted to dithiocarbamate over the reaction of carbon disulfide. Study of the synthesized support in BSA extraction was achieved in further experiments.

β-Carotene is a yellow–orange–red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for β-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding β-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest β-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Fe3O4@C core-shell nanoparticles were modified by (3-aminopropyl)triethoxysilane (APTES) to generate amine functionality in the surface. Then, the amine functional groups were converted to dithiocarbamate via post-modification with carbon disulfide. This nanostructure with new functional property was used to immobilize lipase (obtained from Yarrowia lipolytica U6). Biocatalytic activity of the Fe3O4@C-NHCS-LIP was studied in this project. The interaction of lipase and support though dithiocarbamate binder was examined in the hydrolysis of p-nitrophenyl laurate. In this paper, support showed a unique feature in the immobilization of lipase by maintaining the lipase activity, raising the stability of lipase, and reusability.

Microbial biosynthesis has provided an interesting way to the sustainable production of biofuels. Many efforts have been done to develop efficient microbial platforms to biofuel production in terms of titer and productivity. The oleaginous yeast Yarrowia lipolytica can grow on hydrophobic wastes and accumulate high quantities of lipids as biocrude and fuel feedstock. On the one hand, Y. lipolytica consumes low-cost hydrophobic substrates and reduce their environmental pollution; on the other hand, it produces biotechnologically valuable products such as different lipases and fatty biocrudes which they use in biofuel production. Hence, Y. lipolytica is a promising candidate for sustainable production of fatty acid-based biofuels. Furthermore, new techniques of metabolic engineering and synthetic biology have been used to create Y. lipolytica strains with significantly improved capabilities for biofuel production. The potentials of Y. lipolytica in biofuel production will be reviewed from application of its enzymes in biodiesel manufacture to accumulation of lipids for fatty acid-based advanced biofuels synthesis as well as recent developments in metabolic engineering to make stable and robust strains.

Carbon nanotubes act as regulators of plant germination and growth and are able to change the morphology and physiology of plant cells. The castor plant (Ricinus communis L.) belongs to the Euphorbiaceae family and is a very important medicinal plant. The aim of this study was to investigate the effect of 10 different concentrations of multi-walled carbon nanotubes (MWCNTs) (2, 5, 10, 20, 50, 75, 100, 125, 250 and 500 μg · ml−1) alongside the control under laboratory conditions on the germination and growth of castor seedlings. The results demonstrated that the maximum percentage of germination (96.7%) and relative germination percentage (100%) were found in the concentrations of 50 and 100 μg · ml−1, respectively, and the highest germination rate (53.3%) and the mean germination time (4.6 days) was seen in the concentration of 75 μg · ml−1. However, no statistically significant differences were found between the different concentrations in any of the germination factors. In the concentration of 100 μg · ml−1, there was a significant increase in the seedling vigor index I (400) when compared with the concentrations of 5 and 10 μg · ml−1. The maximum seedling vigor index II (11.3) was found in the concentration of 100 μg · ml−1 and was significantly different from the control and all applied concentrations. The length of radicle in the 100 and 125 μg · ml−1 had a significant increase when compared with the control and the concentrations of 10 and 50 μg · ml−1. The maximum seedling length (4.6 cm) was seen in the concentration of 100 μg · ml−1 where there was a significant increase with 10 μg · ml−1. Moreover, in the 100 μg · ml−1 concentration, the largest number of rootlets (8.6) was seen and when compared with the control and concentrations of 5, 10 and 50 μg · ml−1, there was a statistically significant increase. The maximum wet weight (0.3 g) and dry weight (0.1 g) of seedlings were obtained in the concentration of 100 μg · ml−1 and when compared with the control, there was a significant increase. It was found that in all factors related to the growth of seedlings, the concentrations of 10 and 50 MWCNTs had an inhibitory effect on the response index. The MWCNTs concentration of 100 μg · ml−1 was considered as the optimum concentration in the growth stage of castor seedlings.

Background:Polycyclic aromatic hydrocarbons are a large group of oil contaminants with carcinogenic, mutagenic and teratogenic effects. The release of these compounds in soil destroys animals, plants and microbial diversity and has several negative impacts on physical properties of the soil including the destruction of soil aggregates reduction in pores, and increase in soil bulk density. Many strains of microorganisms isolated have the phenanthrene-degrading ability but this study focused on isolation and identification of a phenanthrene-degrader bacterium for bioremediation of contaminated soils. Background: Polycyclic aromatic hydrocarbons are a large group of oil contaminants with carcinogenic, mutagenic and teratogenic effects. The release of these compounds in soil destroys animals, plants and microbial diversity and has several negative impacts on physical properties of the soil including the destruction of soil aggregates reduction in pores, and increase in soil bulk density. Many strains of microorganisms isolated have the phenanthrene-degrading ability but this study focused on isolation and identification of a phenanthrene-degrader bacterium for bioremediation of contaminated soils. Materials and Methods: Enrichment technique was used for isolation and the most effective isolates, were named pseudomonas aeruginosa ZF1 and Serratia marcescens ZF2. The degradation experiments were conducted in the mineral salt medium (MSM) containing phenanthrene as the sole source of carbon and energy. The selection was based on phenanthrene biodegradation abilities. The isolates were identified using morphological, biochemical tests and 16S rDNA sequencing and after 10 days’ incubation at 30 °C and pH = 7, the bacterial growth and Phe-degrading rate were evaluated by protein assay (Bradford) and gas chromatography (GC), respectively. Results: Biochemical tests and 16s rDNA gene sequence analysis revealed that isolated bacteria are similar to Pseudomonas aeruginosa ZF1 and Serratia marcescens ZF2 with 99% similarity. The results showed a mixture of ZF1 and ZF2 bacteria could degrade 83% at minimum concentrations of 200 ppm of phenanthrene whereas single strain culture of two bacteria had poor degradation abilities (less than 15%). Conclusion:Results showed that isolated co-culture bacteria have high potential to degrade phenanthrene with the best results achieved when the enriched consortium was used and this mixture was shown to be an appropriate candidate for bioremediation purposes.

l-Asparaginase (l-asparagine amidohydrolase; E.C.3.5.1.1) as an anticancer agent is used to treat acute lymphocytic leukemia (ALL), Human Burkitt's lymphoma and non-Hodgkin's lymphoma. The commercial asparaginases are obtained from bacteria Erwinia chrysanthemi and Escherichia coli now which had many side effects. In this study, the effects of a novel l-asparaginase from yeast Yarrowia lipolytica was investigated on human ALL and Burkitt's lymphoma cell lines. The l-asparaginase causes metabolic stress, cytotoxicity, and apoptosis due to the arrest of the G0 cell cycle, the activation of caspase-3 and the modulation of mitochondrial membrane integrity. The RT-PCR analysis showed a significant increase in the pro-apoptosis genes such as Bax, Caspase-3, Caspase-8, Caspase-9 and p53 (P

PURPOSE:Lung cancer is the deadliest known cancer in the world, with the highest number of mutations in proto-oncogenes and tumor suppressor genes. Therefore, this study was conducted to determine the status of hotspot regions in DDR2 and KRAS genes for the first time, as well as in TP53 gene, in lung cancer patients within the Iranian population. EXPERIMENTAL DESIGN:The mutations in exon 2 of KRAS, exon 18 of DDR2, and exons 5-6 of TP53 genes were screened in lung cancer samples, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) using PCR and sequencing techniques. RESULTS:Analysis of the KRAS gene showed only a G12C variation in one large cell carcinoma (LCC) patient, whereas variants were not found in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) cases. The Q808H variation in the DDR2 gene was detected in one SCC sample, while no variant was seen in the ADC and LCC subtypes. Variations in the TP53 gene were seen in all NSCLC subtypes, including six ADC (13.63%), seven SCC (15.9%) and two LCC (4.54%). Forty-eight variants were found in the TP53 gene. Of these, 15 variants were found in coding regions V147A, V157F, Q167Q, D186G, H193R, T211T, F212L and P222P, 33 variants in intronic regions rs1625895 (HGVS: c.672+62A>G), rs766856111 (HGVS: c.672+6G>A) and two new variants (c.560-12A>G and c.672+86T>C). CONCLUSIONS:In conclusion, KRAS, DDR2, and TP53 variants were detected in 2%, 2.17% and 79.54% of all cases, respectively. The frequency of DDR2 mutation is nearly close to other studies, while KRAS and TP53 mutation frequencies are lower and higher than other populations, respectively. Three new putative pathogenic variants, for the first time, have been detected in Iranian patients with lung cancer, including Q808H in DDR2, F212L, and D186G in coding regions of TP53. In addition, we observed five novel benign variants, including Q167Q, P222P and T211T in coding sequence, and c.560-12A>G and c.672+86T>C, in intronic region of TP53. Mutations of KRAS and DDR2 were found in LCC and SCC subtypes, respectively, whereas mutations of TP53 were seen in SCC and ADC subtypes with higher frequencies and LCC subtype with lower frequency. Therefore, Iranian lung cancer patients can benefit from mutational analysis before starting the conventional treatment. A better understanding of the biology of these genes and their mutations will be critical for developing future targeted therapies.

Immobilization of enzymes is a promising approach for the cost-effective application of enzymes. Among others, noncovalent but unleachable approaches for immobilization are one of the most favorable and crucial approaches. Herein, silica nanoparticles are modified by (3-aminopropyl)triethoxysilane (APTES) to generate amino-functionalized silica nanoparticles. Then, the amine functionalities are converted to bifunctional amino acid via post-modification that has zwitterionic properties. This nanostructure with the new functional theme is employed to immobilize Yarrowia lipolytica lipase at room temperature with no further post-modification or cross-linking. This immobilization method is further compared with the metal chelate-based immobilization approach on the same support. The biocatalytic activity of the immobilized lipase is examined under various conditions. The encapsulation of lipase through amino acid-functionalized silica nanoparticles exhibited enhanced stability for the immobilized lipase at higher temperatures and unneutral pHs.

Background and aims. Triple antibiotic paste (TAP) is widely used in endodontics for root canal disinfection, particu-larly in regenerative procedures. The aim of this in vitro study was to evaluate the antimicrobial effects of different concen-trations of TAP at 1-, 2-, 3-, and 4-week intervals on mature Enterococcus faecalis biofilm. Materials and methods. A total of 287 extracted one-rooted human central incisors were infected with E. faecalis ATCC 29212 after removing the crown and preparation. The root canal space was filled with one of the 0.01-, 0.1-, 1-, 10-, 100-, and 1000-mg/mL concentrations of TAP or normal saline (control). The root canal dentin was sampled after 1, 2, 3, and 4 weeks. The dentinal shavings were cultured on Mueller-Hinton agar plates after serial dilutions. The classic colony-forming unit (CFU) counting technique was used to determine remaining bacterial counts. Data were analyzed by using the two-way ANOVA, post hoc Tukey tests and one-way ANOVA (P

Human galanin receptor subtypes GALR1, GALR2, and GALR3 are encoded by separate genes that are located on human chromosomes 18q23, 17q25.3, and 22q13.1, respectively. The exon:intron organization of the gene encoding GALR2 (GALNR2) and GALR3 (GALNR3) is conserved, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 3 and exon 2 encoding the remainder of the receptor, from the second intracellular loop to the COOH-terminus. This conservation of structural organization is indicative of a common evolutionary origin for GALNR2 and GALNR3. The exon:intron organization of the gene encoding GALR1 (GALNR1) is different from that of GALNR2 and GALNR3, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 5, exon 2 encoding the third intracellular loop, and exon 3 encoding the remainder of the receptor, from transmembrane domain 6 to the COOH-terminus. The structural organization of GALNR1 suggests convergent evolution for this gene and represents a structural organization that is unique among genes encoding G-protein-coupled receptors.

Neuropeptide Y is a 36-amino-acid peptide amide with numerous biological activities. These functions are mediated through several pharmacologically distinct receptors. To date five receptor subtypes have been cloned. Here we report the isolation, by low stringency homology cloning from a hypothalamic library, of a cDNA encoding the human homolog of the murine neuropeptide Y receptor subsequently reported (1). Translation of the human Y1-like receptor clone suggested that it encoded a receptor which is truncated in the third extracellular loop. Comparison of the human Y1-like sequence to that of the human Y1 receptor suggested that the truncated receptor could have resulted from a frameshift due to a single nucleotide deletion in the sixth transmembrane domain. Southern blot analysis suggested that the gene is single copy in the human genome. The gene is located on chromosome 5q. To test the hypothesis that allelic variation of nucleic acid length within the sixth transmembrane domain of the Y1-like receptor may exist to produce a functional receptor, genomic DNA from 192 individuals of various ages, ethnic backgrounds, and degrees of obesity were analyzed electrophoretically and by direct sequencing. No variation was detected in any of the subjects, indicating that this receptor subtype may be a transcribed pseudogene in humans.

The melanocortin (MC) peptides mediate a diverse spectrum of biological activities in both the central nervous system and peripheral tissues by interacting with specific guanine nucleotide binding (G protein)-coupled receptors. Previously, four human melanocortin receptor subtypes have been cloned and characterized. In this study, we have isolated mouse complementary DNA (cDNA) and human genomic clones encoding a fifth melanocortin receptor subtype, MC5. Melanocortin peptide stimulation of human MC5, transiently expressed in COS1 cells, results in activation of adenylate cyclase with the following rank order of potency: [Nle4, D-Phe7]-alpha-MSH (melanocyte stimulating hormone)ACTH (1-24) (adrenocorticotropic hormone)alpha-MSHbeta-MSHgamma-MSH. Northern blot hybridization, ribonuclease protection, and reverse transcription/polymerase chain reaction assays indicate that mouse MC5 mRNA is most abundant in skeletal muscle and brain. Lower but detectable levels of MC5 mRNA are also found in RT2-2 retinal neuronal cells, lung, testis, spleen, heart, kidney, and liver.

Most seven-transmembrane G-protein-coupled receptors are rapidly internalized after binding agonist, but the general amino acid recognition sequences mediating this phenomenon have not been identified. In this study, components of the gastrin-releasing peptide receptor (GRP-R) regulating internalization were identified. Four GRP-R mutants with stop codons placed at variable distances distal to the putative palmitoylation sites Cys340-341 were transiently expressed in CHOP fibroblasts. A construct with a minimal carboxyl tail deletion, T375, bound and internalized agonist similarly to wild type receptor. Progressively larger truncations of the carboxyl terminus, however, increasingly impaired GRP-R-mediated internalization without altering receptor-agonist affinity. Three additional constructs were created: one with the putative palmitoylation sites replaced with Ala (CC340-341AA), one with the carboxyl-terminal protein kinase C-consensus sequence converted to Ala (TS360-361AA), and one with all Ser and Thr distal to Cys341 converted to Ala, Asn, or Gly (JF1). All constructs bound agonist similarly to wild type receptor. CC340-341AA internalized similarly to native receptor (93 +/- 3% of wild type by 60 min), whereas internalization of TS360-361AA was partially attenuated (64 +/- 2% of wild type by 60 min). JF1, however, internalized as poorly as T346, with only 16 +/- 2% of the wild type receptors internalized by 60 min. To assess G-protein coupling, selected receptor constructs were stably transfected into Balb fibroblasts, and phosphoinositol hydrolysis was determined. The largest GRP-R truncation, T346, increased total inositol phosphates (EC50 = 2.9 +/- 0.9 nM) similarly to wild type receptor (EC50 = 5.1 +/- 2.2 nM), as did CC340-341AA (EC50 = 5.4 +/- 1.5 nM) and TS360-361AA (EC50 = 3.1 +/- 1.2 nM). These data demonstrate that the multiple Ser and Thr located within the GRP-R carboxyl terminus distal to Cys341, including but not limited to those within the protein kinase C-consensus sequence, specifically regulate GRP-R internalization rates independent of receptor-G-protein coupling.

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane domain V (TM-V) of the NMB-R. To identify which amino acid(s) in TM-V of NMB-R contributed to high affinity NMB binding, four additional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids. Three of the mutations, TyrPheLeu220-222-->PheTyrVal, Ile230-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.

The bombesin (BN)-like peptides mediate a diverse spectrum of biological activities and have been implicated as autocrine growth factors in the pathogenesis and progression of some human small cell lung carcinoma tumors. Previously, two mammalian BN-like peptide receptor subtypes, gastrin-releasing peptide receptor and neuromedin-B receptor, have been cloned and characterized. In this study, we have isolated and characterized human genomic and complementary DNA (cDNA) clones encoding a new BN-like peptide receptor subtype, BN receptor subtype 3 (BRS-3). Expression of BRS-3 cDNA in Xenopus oocytes encodes a functional receptor that is specifically activated by BN-like peptides. Chromosome mapping studies indicate that the BRS-3 gene is located on human chromosome X. BRS-3 mRNA expression in rat tissues is limited to secondary spermatocytes in testis. In contrast, BRS-3 mRNA is widely expressed in a panel of human cell lines from all histological types of lung carcinoma. These results suggest a role for BN-like peptides and their receptors in mammalian reproductive physiology and also indicate that BRS-3 could serve as a potential therapeutic target for human lung carcinoma.

Within the last few years, an increasing number of neuropeptide receptors have been structurally and functionally characterized. The analysis of these receptors, along with a precise understanding of their pharmacology and signal transduction pathways, has been facilitated by the use of molecular genetic techniques. This review will highlight some of the techniques currently being used to study neuropeptide receptors. Methods for isolation of complementary DNA clones for receptors, analysis of receptor messenger RNA expression and characterization of cloned receptors expressed in cultured cells are reviewed. In addition, a genetic approach for the analysis of neuropeptide receptor function in the context of a whole mammalian organism is discussed.

The mutation in a vaccinia virus mutant resistant to inhibition by isatin-β-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the Hin dIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpol32. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.

Genetic and biochemical evidence is presented which shows that the product of the vaccinia virus gene 18R is a virion protein. Western blot analysis of virion proteins using anti-18R serum detects a 78,000-Da protein, localized in the virus core. Of five ts mutants which map to gene 18R, two mutants, ts 10 and ts 44, possess thermolabile virions. Temperature shifts performed during single-step growth of ts 44 suggest that precursors required for virion maturation accumulate during nonpermissive infections with ORF 18R mutants and that protein synthesis is required for recovery from nonpermissive condition.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 ( GALNR2 ) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [ 125 I ]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to G α q/11 to stimulate phospholipase C and increase intracellular calcium levels and to G α i/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)–PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

Mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) are regulatory neuropeptides involved in numerous physiologic processes, and have been implicated as autocrine and/or paracrine growth factors in human lung carcinoma. Three structurally and pharmacologically distinct bombesin receptor subtypes have been isolated and characterized: the gastrin releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). The three receptors are structurally related, sharing about 50% amino acid identity. They are members of the G-protein coupled receptor superfamily with a seven predicted transmembrane segment topology characteristic of receptors in this family. The signal transduction pathway for GRP-R and NMB-R involves coupling to a pertussis-toxin insensitive G-protein, activation of phospholipase C (PLC), generation of inositol trisphosphate (IP3), release of intracellular calcium, and activation of protein kinase C. While all three bombesin receptors are activated by bombesin agonists, GRP-R, NMB-R, and BRS-3 have very different affinities for the mammalian bombesin-like peptides GRP and NMB, as well as bombesin receptor antagonists. The three bombesin receptor subtypes are expressed in an overlapping subset of human lung carcinoma cell lines. Any therapeutic strategy based on modulation of bombesin growth responses in human lung carcinoma would be well served to take into account the pharmacologic heterogeneity of the relevant receptors.

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

The gene affected by five previously isolated temperature-sensitive (ts) mutants (ts 10, ts 18, ts 38, ts 39, ts 44) of vaccinia virus strain WR constituting a single "normal" complementation group has been characterized. Marker rescue and DNA sequence analysis show that the five members of the complementation group map in an open reading frame, ORF 18R, which spans the HindIII I-G junction and has the capacity to encode a 77.6-kDa protein. The nucleotide sequence change responsible for temperature sensitivity in each of the five mutants was determined. Two of the mutants, ts 38 and ts 44, have the identical nucleotide change and may therefore be sisters. Northern blot analysis demonstrates that ORF 18R is transcribed at both early and late times during infection. Two distinct early transcripts have been observed which are 5' coterminal and which contain a 518 nucleotide 5' untranslated region. The long early transcript spans the entire 18R gene while the 3' end of the shorter early transcript maps to an early transcription termination signal contained within the 18R coding sequence. The 5' ends of the late transcripts have been mapped to a family of AUG proximal sites using both S1 nuclease and primer extension analysis. Primer extension analysis also identifies additional late 5' ends which map between nucleotides -500 and -1000 relative to the ORF 18R AUG.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G-protein-coupled receptors. Through expression cloning, human and rat GALR1 receptor cDNA clones have previously been isolated and characterized. In this study, we have used homology screening to isolate a rat brain cDNA clone encoding a second galanin receptor subtype, the GALR2 receptor. The isolated cDNA encodes a 372-amino-acid G-protein-coupled receptor that shares 38% overall amino-acid identity with the rat GALR1 receptor. The pharmacological profile of the rat GALR2 receptor is similar to that of the rat GALR1 receptor. The rat GALR2 receptor binds galanin, N-terminal galanin fragments, and the putative galanin receptor antagonists galantide, C7, M35 and M40 with high affinity but it does not bind C-terminal galanin fragments. Galanin increases intracellular inositol phosphate levels in HEK 293 cells expressing the rat GALR2 receptor via a pertussis toxin-insensitive G-protein. The rat GALR2 receptor mRNA is highly expressed in several brain regions, including hypothalamus and hippocampus as well as the anterior pituitary, with lower levels of expression detected in amygdala, and regions of cortex. It is also highly expressed in the GH3 pituitary cell line and in gut tissues, and to a lower extent in spleen, lung, skeletal muscle, heart, kidney, liver and testis. These results suggest that GALR2 receptor mediates galanin's regulation of pituitary hormone secretion and possibly food intake.

A method is described by which an unselected marker can be inserted into the vaccinia virus genome. Cells were infected with defective virus (either isatin-beta-thiosemicarbazone dependent or temperature sensitive) and then cotransformed with a mixture of full-length wild-type genomic vaccinia virus DNA and a cloned vaccinia DNA molecule containing an allele for phosphonoacetic acid resistance. After incubation under conditions which are nonpermissive for the defective virus but which do not select for incorporation of phosphonoacetic acid resistance, the virus yields were assayed for the presence of all markers involved. Phosphonoacetic acid resistance was inserted into an otherwise wild-type genome with an efficiency of 25-33%. This represents an increase in efficiency of 150-to 3000-fold relative to controls. The procedure should be extremely useful for engineering the vaccinia virus genome.