Biomedical subjects

G D Baldridge

Publications and source records attributed to G D Baldridge.

14 recordsLinked to original sources

PubMed1998

Alfalfa (Medicago sativa L.) resistance to the root-lesion nematode, Pratylenchus penetrans: defense-response gene mRNA and isoflavonoid phytoalexin levels in roots.

Alfalfa (Medicago sativa) varieties with antibiosis-based resistance to the root-lesion nematode (Pratylenchus penetrans), a migratory endoparasite of many crops, have been developed by recurrent selection. Individual plants from these varieties that support significantly lower nematode reproduction were identified for molecular and biochemical characterization of defense responses. Before nematode infection, RNA blot analysis revealed 1.3-1.8-fold higher phenylpropanoid pathway mRNA levels in roots of three resistant plants as compared to three susceptible alfalfa plants. The mRNAs encoded the first enzyme in the pathway (phenylalanine ammonia-lyase), the first in the pathway branch for flavonoid biosynthesis (chalcone synthase), a key enzyme in medicarpin biosynthesis (isoflavone reductase) and a key enzyme in the pathway branch for biosynthesis of lignin cell wall precursors (caffeic acid O-methyltransferase). After nematode infection, the mRNAs declined over 48 h in resistant roots but rose in susceptible plants during the first 12 h after-infection and then declined. Acidic beta-1,3-glucanase mRNA levels were initially similar in both root types but accumulated more rapidly in resistant than in susceptible roots after nematode infection. Levels of a class I chitinase mRNA were similar in both root types. Histone H3.2 mRNA levels, initially 1.3-fold higher in resistant roots, declined over 6-12 h to levels found in susceptible roots and remained stable in both root types thereafter. Defense-response gene transcripts in roots of nematode-resistant and susceptible alfalfa plants thus differed both constitutively and in inductive responses to nematode infection. HPLC analysis of isoflavonoid-derived metabolites of the phenylpropanoid pathway revealed similar total constitutive levels, but varying relative proportions and types, in roots of the resistant and susceptible plants. Nematode infection had no effect on isoflavonoid levels. Constitutive levels of the phytoalexin medicarpin were highest in roots of the two most resistant plants. Medicarpin inhibited motility of P. penetrans in vitro.

Acyltransferases↗

PubMed1998

Phylogenetic placement of rickettsiae from the ticks Amblyomma americanum and Ixodes scapularis.

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tick Amblyomma americanum. We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both the rompA gene and the rompB gene) of MOAa and WB-8-2, another rickettsial isolate from A. americanum. To place MOAa and WB-8-2 and two other nonpathogenic isolates (Rickettsia rickettsii Hip2 and Rickettsia montana M5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9 Rickettsia species and 10 Rickettsia strains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related to R. montana. New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined the rompA gene of a nonpathogenic rickettsial symbiont isolated from the tick Ixodes scapularis. In a phylogenetic analysis, the symbiont was placed as the sister to R. montana and its isolates. The relationship of this symbiont to R. montana raised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.

Amino Acid Sequence↗

PubMed1996

Evidence for a DNA homologous pairing activity in nuclear extracts from mosquito cells.

Using a sensitive homologous pairing/DNA strand transfer assay, we detected formation of joint molecules in the presence of nuclear extract from cultured mosquito C7-10 cells in a reaction containing single stranded circular m13 DNA and a linear, double stranded DNA 5'-end-labeled on the strand complementary to a portion of the single-stranded substrate. Joint molecules were detected by the reduced electrophoretic mobility of labeled probe on agarose gels, which indicated that the 5'-end labeled strand of the linear duplex had formed a hybrid with the single-stranded substrate. Characterization of the activity detected initially in crude nuclear extracts provided a basis for a 5-fold enrichment of activity after a two-step KCl elution from heparin-Sepharose. Further purification by preparative electrophoresis yielded a band at approximately 35 kDa, which, when transferred to Immobilon P membrane, specifically bound the labeled, complementary strand probe. Optimal activity of the electroeluted enzyme required both magnesium and ATP and was sensitive to the ratio of single-stranded and double-stranded DNA substrate and to the amount of protein. This homologous pairing activity from mosquito cells is the first such activity to be described from an insect other than Drosophila melanogaster.

Aedes↗

PubMed1995

Promoter utilization in a mosquito ribosomal DNA cistron.

In the mosquito Aedes albopictus, two potential RNA polymerase I promoters that map 531 and 143 nucleotides upstream of the 18S rRNA gene have been defined on the basis of sequence homology with rRNA promoters from other species. Using the polymerase chain reaction, we confirmed that a 717 nucleotide region spanning the upstream (-531) and downstream (-143) promoters is homogeneous in genomic DNA and in cloned DNA. DNA probes representing each of these promoters, as well as upstream "spacer" promoters, exhibited protein-binding activity, and each unlabeled probe was an effective competitor of protein binding with the other probes, suggesting that these potential regulatory sequences interact with a common protein(s). Analysis of precursor ribosomal RNAs accumulated during temperature shock indicated that transcription is initiated primarily at the upstream (-531) promoter. RNAse protection and primer extension analyses confirmed the predominant use of this promoter, both in cultured cells and in mosquito life stages.

Aedes↗

PubMed1995

Evidence for DNA endonuclease activity in nuclear extracts from mosquito cells.

We describe a deoxyribonuclease activity from nuclear protein extracts of cultured Aedes albopictus mosquito cells. The nuclease cleaved linear and circular double-stranded DNA, first generating 3' OH single-stranded nicks followed by second strand cleavage, but had little or no exonucleolytic activity. Detection of this activity was optimal at pH 7.1, in the presence of a divalent cation (Mg2+, Ca2+, Mn2+, Ba2+). In the presence of Mg2+, Zn2+, Hg2+ and Cu2+ inhibited activity, sulfhydryl reagents and ATP had no effect. At physiological temperatures (18-35 degrees C), linear double-stranded DNA probes were preferentially cleaved near sites containing 3-6 consecutive deoxyadenine/thymine base pairs. Results from salt dependency and drug inhibition studies, combined with inspection of DNA sequence, suggested that DNA structure is among the parameters that determine preferred cleavage sites.

Adenosine Triphosphate↗

PubMed1994

Mosquito large subunit ribosomal RNA: simultaneous alignment of primary and secondary structure.

We report the sequence and propose a secondary structure for the cytoplasmic large subunit (5.8S and 28S) ribosomal RNA of the mosquito, Aedes albopictus, in an aligned format that incorporates secondary structure comparisons with Homo sapiens, Drosophila melanogaster, and Escherichia coli ribosomal RNAs. This format facilitates comparison of subtle differences between models, allowing nucleotide by nucleotide analysis at each position of discrepancy. Comparison of the A. albopictus large subunit ribosomal RNA gene with those from other species revealed new compensatory base changes. The aligned format focuses attention to the specific contribution of the A. albopictus sequence by facilitating comparison with the sequence of another dipteran, D. melanogaster. This is the second report of a complete large subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a member of the lower Diptera (Nematocera).

Aedes↗

PubMed1992

Is higher-order structure conserved in eukaryotic ribosomal DNA intergenic spacers?

Computer-based structural analysis of the ribosomal DNA intergenic spacer (IGS) from the mosquito Aedes albopictus revealed a potential to form strong and extensive secondary structures throughout a 4.7-kilobase (kb) region. The predicted stability of secondary structures was particularly high within a 3.15-kb region containing 17 tandem 201 base-pair subrepeats. Similarly strong secondary structure potential was also found when IGS subrepeats were analyzed from 17 phylogenetically diverse eukaryotes, including vertebrates, invertebrates, and plants. Conservation of higher-order structure potential in the IGS region of ribosomal DNA may reflect evolutionary and functional constraints on chromatin organization, transcriptional regulation of the ribosomal RNA genes, and/or transcript processing and stability.

Aedes↗

PubMed1991

Nucleotide sequence of a mosquito 18S ribosomal RNA gene.

We have sequenced an 18S ribosomal RNA gene from the mosquito, Aedes albopictus. Computer alignment of the 1950 nucleotide coding region (56% A + T) with 18S rRNA sequences from two insect and three vertebrate species revealed greater sequence divergence among the insects than among the vertebrates. Sequence alignments showed that variable region V4, which has been considered to be the most poorly conserved domain in the 18S rRNA gene, was better conserved among insects and vertebrates than was the V6 domain.

Aedes↗

PubMed1991

Ribosomal DNA structure in Aedes mosquitoes (Diptera: Culicidae) and their cell lines.

The molecular organization of the ribosomal DNA repeat unit in laboratory colonies of Aedes albopictus (Skuse) and Aedes aegypti (L.) and in continuous cell lines derived from these species was examined using restriction enzyme mapping and Southern blotting techniques. In the rRNA coding regions, restriction enzyme sites were conserved in Ae. aegypti and Ae. albopictus. In particular, sites that had been mapped in rDNA cloned from the Mauritius strain of Ae. albopictus mosquitoes were confirmed in cloned DNA from cultured Ae. alboptictus cells. The intergenic spacer region of the ribosomal DNA repeat unit in Ae. albopictus was longer than that in Ae. aegypti; contained multiple recognition sites for AluI, SstI, PvuI, and XhoI; and was more heterogeneous in DNA from cultured cells than in DNA from inbred laboratory mosquitoes. The rDNA copy number was 3- to 4-fold lower in cell lines from both Ae. albopictus and Ae. aegypti than in the corresponding adult mosquitoes.

Aedes↗

PubMed1990

Heterologous reassortment of bunyaviruses in Aedes triseriatus mosquitoes and transovarial and oral transmission of newly evolved genotypes.

Aedes triseriatus mosquitoes were orally infected with two different California serogroup bunyaviruses (La Crosse and snowshoe hare viruses) and high frequency reassortment occurred in these mosquitoes. Increased viral replication and subsequent gene segment reassortment was noted in the ovaries of mosquitoes that had ingested multiple blood-meals. To determine whether newly generated reassortant viruses could be transmitted transovarially to progeny mosquitoes, adult female mosquitoes were inoculated with the two temperature-sensitive (ts) parental viruses, and allowed to blood-feed and oviposit. Of 58 infected progeny mosquitoes assayed, six (10%) contained non-ts viruses, and three of these transmitted non-ts viruses to a susceptible host. Selected viruses of the non-ts phenotype, which were isolated from mosquitoes and from mice fed upon by the mosquitoes, were demonstrated to be reassortant viruses by oligonucleotide fingerprinting.

Aedes↗

PubMed1989

Genomic stability of La Crosse virus during vertical and horizontal transmission.

We have used ribonuclease T1 oligonucleotide fingerprint analysis to study genomic stability of La Crosse virus (Bunyaviridae) during vertical and horizontal transmission in the laboratory. No RNA genomic changes were detected in vertebrate cell culture-propagated virus isolated (following ingestion and replication) from the natural host, Aedes triseriatus. Genomic changes were not detected during transovarial passage of the virus through two generations of mosquitoes, nor were changes detected in the genomes of virus isolated from suckling mice that had been fed upon by second generation transovarially-infected mosquitoes. These results demonstrate that despite the well-documented phenomena of rapid nucleotide change in RNA virus genomes under various conditions, the La Crosse virus genome can remain stable during transovarial transmission in the insect host and during transfer between the insect and vertebrate hosts. The evolutionary implications of these results are discussed.

Aedes↗

PubMed1986

Ecdysteroid titer and oocyte growth in the northern house mosquito, Culex pipiens L.

In an anautogenous strain of the northern house mosquito, Culex pipiens, the ovaries reached the resting stage (follicle length = 90 microns) three days after adult emergence. Follicle length increased from 90 to 550 microns between 0 and 60 hr after a blood meal. Total ecdysteroids reached a peak at 400 fmol/insect at 36 hr after a blood meal then declined rapidly. The ratio of 20-hydroxyecdysone to ecdysone increased in conjunction with the total ecdysteroid level. Oocyte growth beyond the resting stage and initiation of vitellogenesis was dependent on a head factor which was released within 4-8 min of the start of the blood meal.

Aging↗

PubMed1985

A rapid method for preparing insect microsomes.

The 1000 g supernatant of a tissue homogenate is layered on top of a small (less than 5 ml) sucrose density gradient and centrifuged for 20 min at very high centrifugal forces in a vertical rotor. Microsomes can be recovered rapidly in suspended form from the middle of the gradient, well separated from mitochondria and soluble (cytosolic) components. Applications to cockroach midgut microsomes and mosquito abdominal tissue microsomes are described, and the method is compared to the classical differential centrifugation method. Cytochrome P-450 monooxygenase activities can be measured on microsomes prepared from midgut tissue of 2-3 Diploptera punctata using this method.

Abdomen↗

PubMedDate not supplied

Primary structure of the ribosomal DNA intergenic spacer from the mosquito, Aedes albopictus.

We have determined the primary structure of a 4.7-kb portion of the ribosomal DNA intergenic spacer from cultured cells of the mosquito, Aedes albopictus. Immediately upstream from the 18S rRNA gene was a 753-bp sequence containing two regions similar to known RNA polymerase I promoters, each preceded by potential transcription termination signals. Upstream from this putative promoter region was a 3.15-kb tandem array of 17 direct repeats with a consensus sequence length of 201 bp. The 201-bp repeats contained imperfect antisense duplications of 11-bp core domain regions in the putative RNA polymerase I promoters, and sequences of possible significance in recombination. Farthest upstream of the 18S rRNA gene was an 803-bp region containing two copies each of 34-, 48-, and 64-bp elements separated by apparently unique sequence. This first detailed structural analysis of a ribosomal DNA intergenic spacer from a member of the lower Diptera has revealed features similar to those described for the higher Diptera as well as conserved motifs presumably critical to rRNA transcription.

Aedes↗