Daniel Suh | PubMed Health

PubMed2003

Mapping of determinants required for the function of the HIV-1 env nuclear retention sequence.

Control of HIV-1 RNA processing and transport are critical to the successful replication of the virus. In previous work, we identified a region within the HIV-1 env that is involved in mediating nuclear retention of unspliced viral RNA. To define this sequence further and identify elements required for function, deletion mutagenesis was carried out. Progressive 5' and 3' deletions map the nuclear retention sequence (NRS) within the intron between nts 8281 and 8381. While deletion of sequences comprising the 3'ss had no effect, removal of the 5'ss resulted in cytoplasmic accumulation of unspliced RNA. Sequence analysis determined that the region corresponding to the NRS is highly conserved among HIV-1 strains. To evaluate whether this NRS interacts with cellular factors, RNA electrophoretic mobility shift assays (REMSA) were performed. We show that the NRS specifically interacts with cellular factors present in HeLa nuclear extracts, and, by UV crosslinking, correlates with the binding of a 49-kDa protein. Immunoprecipitation of the UV crosslinked products determined that this 49-kDa protein corresponds to hnRNP C.

Cell Nucleus↗

PubMed2002

SLC7A9 mutations in all three cystinuria subtypes.

BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acid transport in kidney. Subtypes are defined by the urinary cystine excretion patterns of the obligate heterozygous parents: Type I/N (fully recessive or silent); Type II/N (high excretor); Type III/N (moderate excretor). The first gene implicated in cystinuria (SLC3A1) is associated with the Type I urinary phenotype. A second cystinuria gene (SLC7A9) was recently isolated, and mutations of this gene were associated with dominant (non-Type I) cystinuria alleles. Here we report genotype-phenotype studies of SLC7A9 mutations in a cohort of well-characterized cystinuria probands and their family members. METHODS: Individual exons of the SLC7A9 gene were screened by single strand conformation polymorphism (SSCP) analysis and sequencing of abnormally migrating fragments. RESULTS: Seven mutations were identified. A single bp insertion (799insA) was present in four patients: on Type III alleles in two patients and on Type II alleles in two patients. These results suggest that Type II and Type III may be caused by the same mutation and, therefore, other factors must influence urinary cystine excretion. A 4bp deletion in intron 12 (IVS12+4delAGTA) and a missense mutation (1245G-->A, A354T) were identified on Type III alleles. A nonsense codon (1491G-->T, E436X) and a possible splicing mutation (IVS9-17G-->A) were seen in a Type I/III patient, but the mutations could not be assigned to particular alleles. Of additional interest were two missense mutations (316T-->C, I44T and 967C-->T, P261L) linked to Type I alleles. CONCLUSION: Our results provide evidence that some SLC7A9 mutations may be associated with fully recessive (Type I) forms of cystinuria. We also demonstrate SLC7A9 mutations in dominant Types II and III cystinuria. The finding of SLC7A9 mutations in all three subtypes underscores the complex interactions between specific cystinuria genes and other factors influencing cystine excretion. A simpler phenotypic classification scheme (recessive and dominant) for cystinuria is warranted.

Adult↗