Biomedical subjects

R H Hice

Publications and source records attributed to R H Hice.

6 recordsLinked to original sources

PubMed1997

Organization and molecular characterization of genes in the polyhedrin region of the Anagrapha falcifera multinucleocapsid NPV.

A multinucleocapsid nuclear polyhedrosis virus (MNPV) isolated from the celery looper, Anagrapha falcifera, has been proposed as a new virus based on differences in virulence and DNA fragment profiles between this isolate and the Autographa californica MNPV, the MNPV type species. In the present study, we examined the relatedness of the AfMNPV and AcMNPV genomes by (1) Southern hybridization, (2) comparison of their genetic organization in the polyhedrin gene region (AcMNPV EcoRI-I fragment), and (3) comparison of the nucleotide and deduced amino acid sequences of eight viral genes in this region. Both DNAs hybridized strongly to one another in reciprocal hybridization experiments under stringent conditions, and physical mapping showed that gene order was conserved between the two viruses in the polyhedrin gene region, though the ORF 984 and ctl genes were absent from this region in the AfMNPV. Gene and deduced amino acid sequences for p78, protein tyrosine phosphatase, protein kinase, lef-2, and ORFs 327, 453 and 603, showed identity between the two viruses of greater than 91%. The sequences for the gp64 gene, located on a different EcoRI fragment, were also compared and had a nucleotide sequence identity of 97%, and amino acid sequence identity of greater than 98%. The polyhedrin gene showed the least relatedness between the two viruses, with a nucleotide sequence identity of 80%, and a deduced amino acid sequence identity of 90%. Based on these results, we conclude that the AfMNPV should be considered a variant of the AcMNPV. These results also indicate that caution must be used in basing phylogenetic relationships of NPVs on analysis of a single gene, especially the polyhedrin gene, as is the current practice.

Amino Acid Sequence↗

PubMed1994

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.

Amino Acid Sequence↗

PubMed1993

The 1629-bp open reading frame of the Autographa californica multinucleocapsid nuclear polyhedrosis virus encodes a virion structural protein.

A 1629-bp open reading frame (ORF) of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) is shown to encode a 78-kDa virion structural protein. To determine this, polyclonal antibody was made to a fusion protein synthesized in Escherichia coli from a chimeric gene that contained 1415 bp of the 1629-bp gene. In Western blot analyses, this antibody cross-reacted with a protein of about 78 kDa in both extracellular virions (ECV) and virions isolated from polyhedra (PDV), and with a 78-kDa protein in PDV envelope preparations, but not with PDV nucleocapsids. This suggests that the protein encoded by the 1629-bp ORF is a virion envelope protein or a protein that occurs in the virion intermediate layer between the envelope and nucleocapsid.

Antibodies, Viral↗

PubMed1990

Developmental expression, synthesis, and secretion of insecticyanin by the epidermis of the tobacco hornworm, Manduca sexta.

Insecticyanin was found to be synthesized in several isoelectric forms and stored in the pigment granules in the epidermis. Both major epidermal forms (INS-a, pl 5.5; INS-b, pl 5.7) were found in the cuticle, but only the most basic form, INS-b, was present in the hemolymph. In vitro the epidermis synthesized and secreted both forms into both the cuticle and the medium. Isolation of two cDNA clones for insecticyanin followed by hybridization to epidermal mRNA showed the presence of only one 1.1 kb mRNA, but transcription of the longer cDNA yielded a RNA which produced INS-a but no INS-b. Insecticyanin mRNA was present during the intermolt feeding stages of the 4th and 5th instars and absent during the larval molt and after the onset of metamorphosis. Exposure of either day 2 4th-instar or day 1 5th-instar larval epidermis to 20-hydroxyecdysone (20HE) in vitro caused a dose-dependent decline in this mRNA that was not prevented by simultaneous exposure to JH. When synthesis resumes just before ecdysis, INS-b appears before INS-a; then on the final day of feeding, synthesis of INS-a ceases before that of INS-b. Culture experiments showed that exposure of day 15th epidermis to a small pulse of 20HE followed by an ecdysteroid-free period was best to mimic the in vivo situation, indicating that the small rise of ecdysteroid in the absence of JH on day-2 was responsible for this differential cessation.

Animals↗

PubMed1986

Developmental expression of three genes for larval cuticular proteins of the tobacco hornworm, Manduca sexta.

Three cDNA clones coding for the 12.8, 13.3, and 14.6 kDa larval cuticular proteins of the tobacco hornworm, Manduca sexta, were isolated and characterized. Hybridization to abdominal epidermal RNA from different stages showed that the genes for the 12.8 and 13.3 kDa proteins were expressed only during larval life. By contrast, the gene for the 14.6 kDa protein was expressed throughout the segment during the feeding, growing larval stages, then only in the flexible intersegmental regions during the deposition of endocuticle in the pharate pupa and adult. Quantitative RNA dot blot hybridizations showed that the RNA for each protein disappeared during the larval molt when the ecdysteroid titer was high, then reappeared during the preecdysial deposition of endocuticle. All disappeared when the epidermis became pupally committed at the onset of wandering. Exposure of the fourth instar epidermis to 20-hydroxyecdysone (20HE) in vitro under conditions that lead to the formation of a new larval cuticle by 48 hr caused the disappearance of these RNAs by 18 hr. Exposure of Day 2 fifth instar epidermis to 20HE in vitro caused a depression of these RNAs which in the case of the RNAs coding for the 12.8 and 13.3 kDa proteins was partially prevented by simultaneous exposure to methoprene, a juvenile hormone (JH) mimic. By contrast, the RNA for the 14.6 kDa protein was suppressed by exposure to methoprene alone. Thus, each of these larval cuticular genes is turned off by high ecdysteroid; the presence or absence of JH determines whether or not this suppression is permanent in some or all cells.

Animals↗

PubMed1983

ARS replication during the yeast S phase.

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.

DNA Replication↗