A number of studies have indicated that central nervous system-derived cells can be infected with human immunodeficiency virus type 1 (HIV-1). To determine whether CD4, the receptor for HIV-1 in lymphoid cells, was responsible for infection of neural cells, we characterized infectable human central nervous system tumor lines and primary fetal neural cells and did not detect either CD4 protein or mRNA. We then attempted to block infection with anti-CD4 antibodies known to block infection of lymphoid cells; we noted no effect on any of these cultured cells. The results indicate that CD4 is not the receptor for HIV-1 infection of the glioblastoma line U373-MG, medulloblastoma line MED 217, or primary human fetal neural cells.

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Human immunodeficiency virus type 1 (HIV-1) has been implicated in the generation of acquired immunodeficiency syndrome-associated neurological dysfunction, and it is believed that the presence of CD4 in the nervous system may be involved in the susceptibility of selected neural cell populations to HIV-1 infection. We previously demonstrated (B. Wigdahl, R. A. Guyton, and P. S. Sarin, Virology 159:440 445, 1987) that glial cells derived from human fetal dorsal root ganglion (DRG) are susceptible to HIV-1 infection and subsequently express at least a fraction of the virus genome. In contrast to HIV-1 infection of CD4+ lymphocytes, which can be blocked by treatment with monoclonal antibodies directed against the HIV-1-binding region of CD4 (T4A epitope), treatment of human fetal DRG glial cells with similar antibodies resulted in only a slight reduction in HIV-l-specific gag antigen expression. In addition, preincubation of the HIV-1 inoculum prior to infection with HIV-l-neutralizing antiserum did not reduce HIV-1 gag antigen expression in these cells. Furthermore, we were unable to detect the synthesis or accumulation of the CD4 molecule in neural cell populations derived from DRG. However, a protected CD4-specific RNA fragment was detected in RNA isolated from human fetal DRG and spinal cord tissue by an RNase protection assay with a CD4-specific antisense RNA probe. RNA blot hybridization analysis of total cellular RNA isolated from human fetal DRG and spinal cord demonstrated specffic hybridization to an RNA species that comigrated with the mature 3.0-kilobase CD4 mRNA as well as two unique CD4 RNA species with relative molecular sizes of approximately 5.3 and 6.7 kilobases. Furthermore, all three CD4-related RNA species were polyadenylated when isolated from human fetal spinal cord tissue. These data suggest that HIV-1 infection of human fetal DRG glial cells may proceed via a mechanism of viral entry independent of the T4A epitope of CD4.

Genetic studies have demonstrated that salivary and pancreatic amylase genes are closely linked in human and mouse. To analyze the arrangement of genes within the amylase cluster, a library of YBR mouse genomic DNA was cloned in the cosmid vector pJB8. Clones containing amylase genes were identified by hybridization with amylase cDNA probes. Salivary and pancreatic amylase genes were isolated on separate cosmid clones, but no overlapping clones were evident from the initial screening. A strategy for the rapid isolation of terminal noncoding fragments from the cosmid clones was developed. By using these terminal fragments for chromosome "walking," a map of 78 kilobases of the amylase gene region was constructed. The salivary and pancreatic amylase genes are present within this region in the same 5'-to-3' orientation, separated by 22 kilobases of genomic DNA. A truncated amylase pseudogene is located 10 kilobases downstream from the pancreatic amylase gene.In the human and mouse genomes, distinct genes encode the a-amylases (EC 3.2.1.1) produced in salivary gland and in pancreas (1-4). The extensive sequence homology between salivary (Amy-1) and pancreatic (Amy-2) amylase cDNAs (>90%) indicates that the two genes, Amy-i and Amy-2, are derived from a common ancestral gene (1, 4). Close linkage ofAmy-i and Amy-2 has been established by genetic analysis of the segregation of electrophoretic variants (3, 5). The offspring of more than 1500 meioses have been studied in crosses between inbred mice without observation of recombination between these genes (6). This data suggested that the intergenic distance between Amy-i and Amy-2 may be less than 150 kilobases (kb). Consistent with their close linkage and sequence homology, the amylase genes appear to have undergone "correction" events during mammalian evolution (4, 7).The present study was undertaken to determine the precise organization of amylase gene copies within the mouse multigene cluster. To facilitate analysis of an extended chromosome region, cloning was carried out in a cosmid vector that accommodates 35-to 45-kb inserts of genomic DNA. We chose mouse strain YBR for these studies because of our interest in the independent regulation of its two active pancreatic amylase genes, Amy-2.1 and Amy-2.2 (3, 8-10). We report here the characterization of a 78-kb portion of the amylase gene region that includes one salivary amylase gene, the Amy-2.1 pancreatic amylase gene, and an apparent amylase pseudogene. A subcloning strategy that facilitates chromosome "walking" in cosmid libraries is also described. MATERIALS AND METHODSGenomic DNA libraries were constructed from partially Mbo I-digested DNA isolated from livers of YBR/Ki mice. Restriction fragments of 35-50 kb were size-selected by centrifugation through a neutral sucrose gradient and inserted into' the BamHI site of the cosmid vector pJB8 (11, 12). Recombinant molecules were packaged in vitro into X phage heads and used to infect Escherichia coli strain 490A (13). The yield of infectious phage particles...