C. Thomas Caskey

Spontaneous reversion of novel Lesch-Nyhan mutation byHPRT gene rearrangement

Molecular analysis of an unusual patient with the Lesch-Nyhan syndrome has suggested that the mutation is due to a partial HPRTgene duplication. We now report the cloning and sequencing of the mutant HPRTcDNA which shows the precise duplication of exons 2 and 3. This mutation is the result of an internal duplication of 16–20 kilobases of the gene. The structure of the mutant gene suggests that the duplication was not generated by a single unequal crossing-over event between two normal HPRTalleles. Growth of Epstein-Barr virus-transformed lymphoblasts from this patient in selective medium has permitted isolation of spontaneous HPRT+revertants of this mutation. The reversion event involves a second major HPRTgene rearrangement where most or all of the duplicated portion of the mutant gene is deleted. The original mutation therefore has the potential for spontaneous somatic reversion. This may explain the relatively mild symptoms of the Lesch-Nyhan syndrome exhibited by this patient.

Alu PCR: The Use of Repeat Sequence Primers for Amplification of Human DNA from Complex Sources

The polymerase chain reaction (PCR) has revolutionized the isolation and analysis of specific nucleic acid fragments from a wide variety of sources.1 However, application of the PCR to isolate and analyze a particular DNA region has required knowledge of DNA sequences flanking the region of interest. This limits amplification to regions of known DNA sequence. We sought to amplify human DNA of unknown sequence from complex mixtures of human and other species DNAs. In particular, we have applied the PCR to isolation of human DNA specifically from somatic cell hybrids retaining human chromosome fragments in rodent cell backgrounds. This allows the isolation and characterization of sequences from specific human regions retained in hybrids, obviating the requirement of cloned DNA libraries and isolation of human clones through the use of human-specific repeat sequence probes.2 The method, Alu PCR, has also proven useful for the rapid isolation of human insert DNA from cloned sources as well, extending the application of the PCR to genomic DNAs cloned in lambda and yeast artificial chromosome (YAC)3 vectors. The adaptation of the PCR to large genomic regions provides another tool for the analysis of the human genome.

Gene therapy : a new approach for the treatment of genetic disorders

Clinical Pharmacology and Therapeutics (1990) 47, 1–11; doi:10.1038/clpt.1990.1

Generation of cDNA Probes by Reverse Translation of Amino Acid Sequence

A common approach to cDNA clone isolation involves the use of a consensus oligonucleotide probe corresponding to the protein amino acid sequence (1). A correct selection of the oligonucleotide probe is difficult since the genetic code is degenerate (2). Oligonucleotide probes lacking complementarity to their target sequences generate spurious hybridization signals and fail to identify authentic clones, rendering cDNA library screening a complicated and frequently an unproductive process (3,4). Strategies designed to overcome these problems involve the selection of multiple oligonucleotide probes, inclusion of the most frequently used codons (5–7) and replacement of ambiguous nucleotides with deoxyinosine and 5-fluorodeoxyuridine (8,9). These approaches are frequently unsatisfactory since actual codon use in the target sequence is quite heterogeneous, thus lowering the likelihood of a perfect match with the synthetic oligonucleotide probe.

A genome approach to the human X chromosome

The human X chromosome includes many disease genes, some of which have already been cloned using time-consuming and labor-intensive methods. A more efficient way to study this chromosome makes use of technology emerging from the human genome initiative.