Extensive variation of human immunodeficiency virus type-1 in vivo

Genotypic variation among independent isolates of human immunodeficiency virus type-1 (HIV-1) is well known, but its molecular basis and biological consequences are poorly understood. We examined the genesis of molecular variation in HIV-1 by sequential virus isolations from two chronically infected individuals and analysis of recombinant HIV-1 genomic clones. In three different virus isolates full-length HIV-1 clones were identified and found to consist, respectively, of 17, 9 and 13 distinguishable, but highly-related, viral genotypes. Thirty-five viral clones derived from two HIV-1 isolates obtained from the same individual but 16 months apart showed progressive change, yet were clearly related. Similar changes in the HIV-1 genome did not occur in vitro during virus isolation and amplification. The results indicate that HIV-1 variation in vivo is rapid, that a remarkably large number of related but distinguishable genotypic variants evolve in parallel and coexist during chronic infection, and that 'isolates' of HIV-1, unless molecularly or biologically cloned, generally consist of complex mixtures of genotypically distinguishable viruses.     

A point mutation of the rhodopsin gene in one form of retinitis pigmentosa

The gene for autosomal dominant retinitis pigmentosa in a large pedigree of Irish origin has recently been found to be linked to an anonymous polymorphic sequence, D3S47 (C17), from the long arm of chromosome 3. As the gene coding for rhodopsin is also assigned to the long arm of chromosome 3 and is expressed in rod photoreceptors that are affected early in this blinding disease, we searched for a mutation of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. We found a C----A transversion in codon 23 (corresponding to a proline----histidine substitution) in 17 of 148 unrelated patients and not in any of 102 unaffected individuals. This result, coupled with the fact that the proline normally present at position 23 is highly conserved among the opsins and related G-protein receptors, indicates that this mutation could be the cause of one form of autosomal dominant retinitis pigmentosa.     

Current applications of single-cell PCR

The advent of the polymerase chain reaction (PCR) has revolutionised the way in which molecular biologists view their task at hand, for it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell. It is especially in the field of prenatal diagnostics that this ability has been readily seized upon, as it has opened up the prospect of preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. However, apart from diagnostic applications, single-cell PCR has proven to be of enormous use to basic scientists, addressing diverse immunological, neurological and developmental questions, where both the genome but also messenger RNA expression patterns were examined. Furthermore, recent advances, such as optimised whole genome amplification (WGA) procedures, single-cell complementary DNA arrays and perhaps even single-cell comparative genomic hybridisation will ensure that the genetic analysis of single cells will become common practice, thereby opening up new possibilities for diagnosis and research.