Linkage disequilibrium mapping of the gene for Hermansky-Pudlak syndrome to chromosome 10q23. 1-q23.3

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by the triad of tyrosinase-positive oculocutaneous albinism, bleeding diathesis due to storage-pool deficiency of platelets, and a lysosomal ceroid storage disease. The disorder is particularly frequent in Puerto Rico and in an isolated village in the Swiss Alps. We have used a linkage disequilibrium mapping approach to localize the HPS gene in both of these groups to a 0.6 centiMorgan interval in chromosome segment 10q23.1-q23.3. These results indicate that the Puerto Rican and Swiss forms of HPS are either allelic or that they result from mutations in very closely linked genes in this region. This region of distal chromosome 10q is syntenic to the region of mouse chromosome 19 that includes ‘pale ear' (ep) and ‘ruby-eye' (ru), which must be considered as potential murine homologues to human HP

Organization and Nucleotide Sequence of the Human Hermansky-Pudlak Syndrome (HPS) Gene

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, bleeding tendency, and lysosomal ceroid storage disease, associated with defects of multiple cytoplasmic organelles-melanosomes, platelet-dense granules, and lysosomes. HPS is frequently fatal and is the most common single-gene disorder in Puerto Rico. We previously characterized the human HPS cDNA and identified pathologic mutations in the gene in patients with HPS. The HPS protein is a novel apparent transmembrane polypeptide that seems to be crucial for normal organellar development. Here we describe the structural organization, nucleotide sequence, and polymorphisms of the human HPS gene. The gene consists of 20 exons spanning about 30.5kb in chromosome segment l0q23.1-q23.3. One of the intervening sequences is a member of the novel, very rare class of so-called “AT-AC” introns, defined by highly atypical 5' and 3' splice site and branch site consensus sequences that provide novel targets for possible pathologic gene mutations. This information provides the basis for molecular analyses of patients with HPS and will greatly facilitate diagnosis and carrier detection of this severe disorder

Mutations in the U5 Sequences Adjacent to the Primer Binding Site Do Not Affect tRNA Cleavage by Rous Sarcoma Virus RNase H but Do Cause Aberrant Integrations In Vivo

In most retroviruses, the first nucleotide added to the tRNA primer becomes the right end of the U5 region in the right long terminal repeat (LTR); the removal of this tRNA primer by RNase H defines the right end of the linear double-stranded DNA. Most retroviruses have two nucleotides between the 5′ end of the primer binding site (PBS) and the CA dinucleotide that will become the end of the integrated provirus. However, human immunodeficiency virus type 1 (HIV-1) has only one nucleotide at this position, and HIV-2 has three nucleotides. We changed the two nucleotides (TT) between the PBS and the CA dinucleotide of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z to match the HIV-1 sequence (G) and the HIV-2 sequence (GGT), and we changed the CA dinucleotide to TC. In all three mutants, RNase H removes the entire tRNA primer. Sequence analysis of RSVP(HIV2) proviruses suggests that RSV integrase can remove three nucleotides from the U5 LTR terminus of the linear viral DNA during integration, although this mutation significantly reduced virus titer, suggesting that removing three nucleotides is inefficient. However, the results obtained with RSVP(HIV1) and RSVP(CATC) show that RSV integrase can process and integrate the normal U3 LTR terminus of a linear DNA independently of an aberrant U5 LTR terminus. The aberrant end can then be joined to the host DNA by unusual processes that do not involve the conserved CA dinucleotide. These unusual events generate either large duplications or, less frequently, deletions in the host genomic DNA instead of the normal 5- to 6-base duplications

Integration of Rous Sarcoma Virus DNA: a CA Dinucleotide Is Not Required for Integration of the U3 End of Viral DNA▿ †

The two ends of RSV linear DNA are independently inserted into host DNA by integrase in vivo. We previously showed that the range of U3 sequences that are acceptable substrates for integrase appeared to be greater than the range of acceptable U5 sequences in vivo. We have done additional experiments to determine which U3 sequences are good integrase substrates. On the U3 end, there does not appear to be a stringent requirement for the canonical CA, integrase can efficiently remove three nucleotides, and six nucleotides are sufficient to allow integration with reasonable, albeit reduced, efficiency

Construction and Characterization of a Replication-Competent Retroviral Shuttle Vector Plasmid

We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs