Claudia A. Sutton

New heat shock puffs and β=galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene

A hybrid gene that consists of the Drosophila heat shock gene, hsp70, fused to the E. coli beta-galactosidase gene has been introduced into the Drosophila germline by the P element microinjection method. This hybrid includes 194 bp of sequence upstream of the start of the hsp70 transcript. Three strains of transformed flies were isolated and characterized by DNA blotting experiments and by in situ hybridization to polytene chromosomes. Strain Bg61 has a single insert of the hybrid gene at the tip of chromosome 3L, site 61A, and the insert consists of a structure that is consistent with P-element-mediated transposition. Strain Bg9,61 has inserts at both 61A and 9E, while Bg64 has a single insert at 64D. Heat shock induces the formation of a large chromosomal puff at all three sites. These puffs appear and regress with kinetics indistinguishable from the puffing of the heat shock locus, 87C, from which the hsp70 gene, used in these studies, was isolated. The beta-galactosidase activity in the transformants is inducible by heat shock and shows a widespread distribution throughout the tissues of larvae and adults.

Plant organelle gene expression: Altered by RNA editing

Transcripts of the higher plant plastid and mitochondrial genomes undergo cytosine-to-uracil RNA editing. Although only about 0.13% of the codons in the entire maize chloroplast genome are altered, plant mitochondrial protein coding genes typically have 3–15% of the codons affected by editing. Incompletely edited transcripts of certain genes are abundant, indicating that plant organelle-transcript editing is a post-transcriptional process. Partially edited transcripts can enter the translation apparatus, resulting in more than one protein product encoded by the same gene. How are cytosine residues selected for editing? Recent data from chloroplast transformants and abnormal mitochondrial genes provide some clues.

Editing of pre-mRNAs can occur before cis- and trans-splicing in Petunia mitochondria.

Plant mitochondrial mRNAs have recently been shown to undergo editing, involving cytidine-to-uridine changes relative to the DNA sequence. We have examined the temporal relationship of editing and intron removal in coxII mRNAs in Petunia mitochondria. By using differential hybridization to probes specific for edited and unedited RNA and by sequencing of individual unspliced coxII pre-mRNA cDNAs, we found that RNA editing at any editing site can precede the splicing event. Similar results were obtained from examinations of pre-mRNA cDNAs of nad1, a gene composed of multiple exons that are both cis and trans spliced. Thus, intron removal is not required before editing can occur. The existence of editing intermediates indicates that the editing process is not strictly coincident with transcription.

Genomic Variation of the Fibropapilloma-Associated Marine Turtle Herpesvirus across Seven Geographic Areas and Three Host Species

ABSTRACT Fibropapillomatosis (FP) of marine turtles is an emerging neoplastic disease associated with infection by a novel turtle herpesvirus, fibropapilloma-associated turtle herpesvirus (FPTHV). This report presents 23 kb of the genome of an FPTHV infecting a Hawaiian green turtle (Chelonia mydas). By sequence homology, the open reading frames in this contig correspond to herpes simplex virus genes UL23 through UL36. The order, orientation, and homology of these putative genes indicate that FPTHV is a member of the Alphaherpesvirinae. The UL27-, UL30-, and UL34-homologous open reading frames from FPTHVs infecting nine FP-affected marine turtles from seven geographic areas and three turtle species (C. mydas, Caretta caretta, and Lepidochelys olivacea) were compared. A high degree of nucleotide sequence conservation was found among these virus variants. However, geographic variations were also found: the FPTHVs examined here form four groups, corresponding to the Atlantic Ocean, West pacific, mid-Pacific, and east Pacific. Our results indicate that FPTHV was established in marine turtle populations prior to the emergence of FP as it is currently known.

Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma.

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B cells: 21 chronic lymphocytic leukemia (B-CLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients (B-ALL, B-CLL or AML, but not MM) was positive for EBV and contained <100 ebv copies/100 ng dna. ebv loads were occasionally high (>500 copies/100 ng DNA) in B-ALL (2/16) and in B-CLL (2/21) samples. The fractions of samples positive for HHV-8 and HHV-6A, less than 10% for MM patients, were high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV-6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+). B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of multiple HHVs, including EBV and CMV at high loads, is not rare in B-ALL and B-CLL cell samples. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.

Identification and Characterization of an Exogenous Retrovirus from Atlantic Salmon Swim Bladder Sarcomas

ABSTRACT A novel piscine retrovirus has been identified in association with an outbreak of leiomyosarcoma in the swim bladders of Atlantic salmon. The complete nucleotide sequence of the Atlantic salmon swim bladder sarcoma virus (SSSV) provirus is 10.9 kb in length and shares a structure and transcriptional profile similar to those of murine leukemia virus-like simple retroviruses. SSSV appears unique to simple retroviruses by not harboring sequences in the Atlantic salmon genome. Additionally, SSSV differs from other retroviruses in potentially utilizing a methionine tRNA primer binding site. SSSV-associated tumors contain high proviral copy numbers (greater than 30 per cell) and a polyclonal integration pattern. Phylogenetic analysis based on reverse transcriptase places SSSV with zebrafish endogenous retrovirus (ZFERV) between the Gammaretrovirus and Epsilonretrovirus genera. Large regions of continuous homology between SSSV and ZFERV Gag, Pol, and Env suggest that these viruses represent a new group of related piscine retroviruses.

A Plant Mitochondrial Sequence Transcribed in Transgenic Tobacco Chloroplasts Is Not Edited

RNA editing occurs in two higher-plant organelles, chloroplasts and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components.

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco.

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.

Hepatitis C virus, human herpesvirus 8, and the development of plasma-cell leukemia.

To the Editor: The role of hepatitis C virus (HCV) and human herpesvirus 8 (HHV-8), two B-cell–tropic viruses, in B-cell proliferation1 is illustrated by the following unusual case of plasma-cell l...

Localization and expression of transformed DNA sequences within heat shock puffs ofDrosophila melanogaster

In situ hybridization at high resolution with biotin-labeled DNA was used to locate specific transcriptional units within the chromosomal puffs of normal heat shock loci and of new heat shock loci generated by transformation. This method resolves copies of thehsp70 gene that are separated by 40 kb within the 87C heat shock locus. In the case of two new puff loci generated by transformation, the heat-activated transcript is encoded by sequences that reside entirely within the puff domain and the activated promoter is positioned asymmetrically within the puff. However, an adjacent promoter and its transcriptional unit which do not appear to be induced by heat shock are also within the puff. These results support the idea that a chromosomal puff is a structure that facilitates or results from vigorous transcription, but that the structural change alone is insufficient to induce transcription.