Approximately 2,000 embryogenic uninuclear microspores of rapeseed (Brassica napus) cv. Topas were intranuclearly injected with a chimaeric ;3-glucuronidase (Escherichia coli Uid A) gene. Stable integration had not occurred among 55 plants that were regenerated. Coinjection of the dye Lucifer Yellow and detection of injected DNA by the polymerase chain reaction revealed high frequencies of transfer. However, the amount of DNA injected was less than 20 copies, which may have been insufficient for stable transformation of microspores.

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cDNA and genomic clones of a new pollen-specific gene, Bnm1, have been isolated from Brassica napus cv. Topas. The gene contains an open reading frame of 546 bp and a single intron of 362 bp. A comparison of the deduced amino acid sequence with sequences in data banks did not show similarity with known proteins. Northern blot analysis of developing pollen showed that Bnm1 mRNA was first detected in bicellular pollen and accumulated to higher levels in tricellular pollen. Bnm1 mRNA was not detected in leaves, stems, roots, pistils, seeds or pollen-derived embryos. RNA in situ hybridization of whole flower buds confirmed that Bnm1 was pollen-specific and expressed late in development. A promoter fragment of the Bnm1 gene fused to the gusA reporter gene yielded similar patterns of tissue specificity and developmental regulation in transgenic B. napus cv. Westar plants; however, the promoter was also active during the early stages of pollen development. The Bnm1 gene, cloned in this study, was derived from the A genome of the allotetraploid species B. napus (AACC). Southern blot analysis indicated that sequences similar to the Bnm1 gene were found in both A and C Brassica genomes. Related sequences were found in all 10 members of the Brassiceae tribe examined, but were not present in all tribes of the Brassicaceae family.

A reverse transcriptase-PCR strategy was developed for the detection of hog cholera virus. Hog cholera virus template was amplified from tissue culture fluids and from tissues and blood of infected pigs, but not from samples containing other pestiviruses. Restriction endonuclease analysis identified samples as historic or recent isolates. Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV), and border disease virus (BDV) constitute the Pestivirus genus, which has recently been classed as a member of the Flaviviridae (2, 17). Pestiviruses contain a single positivestrand RNA, approximately 13 kb in length, which includes a sole large open reading frame (4, 9, 11). Structural proteins are processed from the 5' end, while nonstructural proteins are found at the 3' end of the polyprotein (3). Pestiviruses are associated with economically important diseases of animals. Acute HCV infection is characterized by high mortality, with pigs exhibiting clinical signs of fever, inappetence, diarrhea, and terminally, purplish discoloration of the extremities (15). Chronic infections involve periods of anorexia, fever, and diarrhea. Infection of pregnant sows may lead to abortion or birth of mummified, stillborn, or weak piglets.

PCR was performed on DNA extracts derived from clinical serum samples submitted for human herpesvirus 6 (HHV-6) serological examination. To detect amplified HHV-6 products, a hybridization-based microtiter plate assay (PCR ELISA; Boehringer Mannheim) was used. The assay system was found to be rapid, specific, and sensitive. Approximately three copies of a plasmid-based HHV-6 sequence could be detected, and no cross amplification was observed with HHV-7 genomic DNA. There was no correlation found between HHV-6 DNA detection and serological status in clinical serum samples from individuals more than 2 years old. On the other hand, in serum samples from infants less than 2 years old, a high rate of detection of HHV-6 DNA was observed in those who lacked immunoglobulin G and M antibodies to HHV-6 (55%). In this regard, PCR of serum DNA extracts may be used as a sensitive indicator of active HHV-6 infection in infants prior to their seroconversion.