DNA and Cell Biology

A 17-kDa, immunodominant antigen of Bartonella henselae Houston-1 has previously been cloned, sequenced, and characterized. This clone (H13) contains the 17-kDa antigen gene plus a partial open reading frame, designated ORF1, which is 459 nucleotides long and is directly upstream of the 17-kDa gene. Comparison of the deduced partial amino acid sequence of ORF1 with that of other known genes in GenBank revealed significant identity with several other bacterial virulence genes, including VirB4 of the Agrobacterium tumefaciens virB operon (56/149 amino acids). An overlapping clone, pGB3, was recovered and shown to contain a 3.0-kb region upstream of the 17-kDa gene. Sequence analysis revealed three ORFs upstream of the gene. The deduced amino acid sequence of each ORF was compared with sequences in GenBank, and identity was found with VirB2, VirB3, and VirB4 of A. tumefaciens. In vitro transcription/translation and SDS-PAGE demonstrated that three proteins of 9 kDa, 10 kDa, and 92 kDa, corresponding to the predicted molecular weight of 10.9 kDa, 11.7 kDa, and 89.9 kDa of VirB2, VirB3, and VirB4, respectively, could be expressed from these coding regions. These results indicate that virulence-associated genes and their overall chromosomal arrangement are relatively well conserved between B. henselae and other gram-negative bacteria such as A. tumefaciens.

The Drosophila S3 protein is known to be associated with ribosomes, where it is thought to play a role in the initiation of protein translation. The S3 protein also contains a DNA repair activity, efficiently processing 8- oxoguanine residues in DNA via an N-glycosylase/apurinic-apyrimidinic (AP) lyase activity. The gene that encodes S3 has previously been localized to one of the Minute loci on chromosome 3 in Drosophila. This study focused on the genomic organization of S3 at M(3)95A, initial promoter characterization, and analysis of three mutant alleles at this locus. The S3 gene was found to be a single-copy gene 2 to 3 kb in length and containing a single intron. The upstream 1.6-kb region was analyzed for promoter activity, identifying a presumptive regulatory domain containing potential enhancer and suppressor elements. This finding is of interest, as the S3 gene is constitutively expressed throughout development and mRNA is most likely maternally inherited. Lastly, three Minute alleles from the same locus were sequenced and two alleles found to contain a 22- bp deletion in exon 2, resulting in a truncated S3 protein, although wildtype levels of S3 mRNA and protein were detected in the viable heterozygous Minute alleles, possibly reflecting dosage compensation.

DNA-raised antibody (Ab) responses have been compared for the dependence on CD4 + and CD8 + cells, the longevity of functional antigen (Ag) expression, and the nature of the Ag-presenting cell after intramuscular(IM) and gene gun inoculations. A plasmid expressing the hemagglutinin (HA) glycoprotein of influenza virus was used for immunizations of BALB/c mice. Intramuscular and gene gun-raised Abs had similar dependencies on CD4 + and CD8 + cells but different temporal patterns of functional Ag expression. The two methods of DNA immunization also appeared to have different frequencies or types of Ag-presenting cells in the draining lymph nodes and spleen. For both methods of DNA delivery, Ab was independent of CD8 + cells but dependent on CD4 + cells. The CD4 dependence occurred at priming but not booster immunizations and resulted in a 1-month delay in the Ab response. Temporal T-cell transfers from TCR +/+ mice into immunized TCR -/- mice revealed the persistence of DNA-expressed Ag for up to 1 month after both IM and gene gun inoculations. For gene gun, but not IM immunizations, approximately 90% of the functional Ag expression was lost by 1 week, consistent with the sloughing of the epidermal target site. Despite similar titers of raised Ab, Ag-presenting dendritic cells could be detected in the draining lymph nodes and spleen of gene gun- but not IM DNA-immunized mice. In the gene gun-immunized mice, Ag-presenting dendritic cells appeared in the draining lymph nodes before the spleen.

The purpose of this study was to identify the cis-acting elements and the trans-acting factors involved in the iron-induced expression of the collagen alpha 1(I) (COL1aI) gene. Rat hepatic stellate cells were cultured in the presence of 50 muM ferric chloride, 50 muM ascorbic acid, and 250 muM citric acid (Fe/AA/CA), and the effects on collagen gene expression and the binding of nuclear proteins to the COL1aI promoter were measured. The Fe/AA/CA treatment induced a time- and dose-dependent increase in the cellular levels of COL1aI mRNA that was abrogate by pretreating cells with cycloheximide, antioxidants, and inhibitors of aldehyde-protein adduct formation. Transient transfection experiments showed that Fe/AA/CA exerted its effect through regulatory elements located between- 220 and - 110 bp of the COL1aI promoter. Gel retardation assays showed that Fe/AA/CA increased the binding of nuclear proteins to two elements located between - 161 and - 110 bp of the COL1aI promoter. These bindings were blocked by unlabeled consensus Sp1 oligonucleotide and supershifted with Sp1 and Sp3 antibodies. Finally, Fe/AA/CA increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Treatment with Fe/AA/CA stimulates COL1aI gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between - 161 and - 110 bp of the COL1aI promoter.

Human immunodeficiency virus type 1 (HIV-1) is known for its ability to infect immune cells, including Tcells and macrophages. The 96-amino acid Vpr, a virion-associated protein, is essential for viral replication in monocytes/macrophages and increases viral replication in primary and established T-cell lines. The Vpr protein regulates a number of host cellular events, including proliferation, differentiation, apoptosis, cytokine production, and NF-kappaB-mediated transcription. Most of these functions have been analyzed using either endogenous Vpr protein or cells transfected with a Vpr expression plasmid. We developed a lentiviral vector complemented with a Vpr expression plasmid that results in viral particles packaged with Vpr protein. To facilitate identification of the target cells infected with the particles containing Vpr, we fused green fluorescent protein (GFP) with the Vpr open reading frame and analyzed the biology of this novel particle. Vpr itself is expressed as a 14-kDa protein; however, in vitro translation of the pVpr-GFP plasmid resulted in the expression of 39-kDa fusion protein. The fusion molecule exhibited the same activity in arresting the cell cycle in G₂ as does the wildtype Vpr molecule. Subcellular localization of Vpr and Vpr-GFP by immunofluoresence in human and murine cell lines indicated that Vpr by itself or with the reporter GFP showed a perinuclear staining pattern. Replication kinetics showed no significant difference between Vpr-GFP and native complemented pseudovirus replication in a single-round infectivity assay. A flow cytometry analysis of peripheral blood lymphocytes and macrophages infected with Vpr-GFP-packaged virions and selected by GFP showed 56.7% infectivity for lymphocytes and 84.6% infectivity for macrophages. Additional analysis of CD24 (HSA)-positive cells showed infection of CD4 + cells, macrophages, and, importantly, dendritic cells. This system will allow us to identify specific cell populations including antigen-presenting cells, and allow quantitative analysis of the precise effect of Vpr on both target and bystander cells in vitro as well as in vivo.

Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have suggested that it is involved in the formation of coated vesicles. In this report, the differential expression of four human dynamin-like protein splice variants (HdynIV-wildtype [WT],-11, -26, and-37) from various brain tumors was identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel variant (HdynIV-11), not described previously, was identified. The four alternatively spliced variants exhibited tissue specificity in normal tissues. The HdynIV-WT was strongly expressed in the brain, whereas HdynIV-37 was expressed in all tissues examined. Moreover, HdynIV-26 was dominant in the liver and apparently overexpressed in all astrocytomas and most meningiomas and adenomas. This report suggests that HdynIV-26 may cause aberrant protein trafficking and alter vesicle formation in brain tumors. Our results also suggest that dynamin-like protein is associated with various brain tumors and, more importantly, that aberrant expression of the HdynIV-26 variant may play a role in brain tumorigenesis.