The latency-associated transcript (LAT) is the major viral transcript detected by in situ hybridization of mouse and human sensory ganglia latently infected with herpes simplex virus type 1. The last 750 bases of LAT are complementary to infected-cell polypeptide 0, a herpes simplex virus type 1 immediate-early gene that encodes a transactivating protein that may facilitate re-activation of the virus from the latent state. Several laboratories have shown that LAT accumulates in the nucleus and is not polyadenylylated. Recently, we showed that the promoter for LAT lies 688 bases upstream from its 5' end. We report here that LAT is actually a uniquely stable intron. Furthermore, LAT effectively inhibits transactivation of gene expression by infected-cell polypeptide 0 in transient transfection assays.

Successful human development is dependent upon a cascade of events following fertilization. Unfortunately, knowledge of these critical events in humans is remarkably incomplete. Although hundreds of thousands of human embryos are cultured yearly at infertility centers worldwide, the vast majority fail to develop in culture or following transfer to the uterus. In this study, we sought to characterize global patterns of gene expression in individual, normal embryos during the first three days of embryonic life using microarrays; we then compared gene expression between normally growing and growth-arrested embryos using quantitative PCR. Our results documented several novel findings. First, we found that a complex pattern of gene expression exists; most genes that are transcriptionally modulated during the first three days following fertilization are not upregulated, as was previously thought, but are downregulated. Second, we observed that the majority of genes exhibiting differential expression during preimplantation development are of unknown identity and/or function. Third, we show that embryonic transcriptional programs are clearly established by day 3 following fertilization, even in embryos that arrested prematurely with 2-, 3- or 4-cells. This indicates that failure to activate transcription is not associated with the majority of human preimplantation embryo loss. Finally, taken together, these results provide the first global analysis of the human preimplantation embryo transcriptome, and demonstrate that RNA can be amplified from single oocytes and embryos for analysis by cDNA microarray technology, thus lending credence to additional studies of genetic regulation in these cell types, as well as in other small biological samples.

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The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species in latently infected mouse neurons. Previous sequence analysis revealed no obvious promoter elements near the 5' end of the LAT, but a TATA box and other potential promoter elements were found 700 base pairs upstream. A recombinant virus in which the rabbit beta-globin gene was inserted immediately downstream of the TATA box expressed globin mRNA and did not express the LAT. A second recombinant virus, in which this TATA box was removed, was negative for LAT expression in a latent infection. The location of the LAT promoter suggested that RNA upstream of the LAT was synthesized and degraded during latent-phase transcription. Low levels of this RNA were observed by in situ hybridization. In other experiments, RNA from a productive infection was used to detect a transcript extending from the LAT promoter to a polyadenylation signal approximately 8.5 kilobases downstream. These data suggest that the LAT may be processed from a larger transcription unit which begins distal to the TATA box 700 base pairs upstream of the LAT and extends to a polyadenylation signal almost 5 kilobases downstream of the 3' end of the LAT.

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 mm 2 , 113 mm 2 , and 86 mm 2 respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.