Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement.

We studied the pattern of BCR involvement in 52 patients with chronic myeloid leukemia by Southern blotting. Of 33 Philadelphia (Ph)-positive patients, 30 had evidence of M-BCR rearrangement, two cases were difficult to interpret, and one clearly lacked evidence of M-BCR rearrangement. Of 19 Ph-negative patients, nine showed M-BCR rearrangement, nine showed no rearrangement, and one result was uncertain. We selected for more detailed study eight patients (three Ph-positive and five Ph-negative). Two of the Ph-positive patients, whose Southern blots were difficult to interpret, had rearranged bands when the BCR gene was studied by pulsed field gel electrophoresis (PFGE). Results of PFGE studies and in situ hybridization to metaphase chromosomes in the third Ph-positive patient, whose DNA clearly lacked M-BCR rearrangement on Southern analysis, were consistent with a breakpoint on chromosome 22 located 3' of all known exons of the BCR gene. However, mRNA studied with the polymerase chain reaction showed evidence of a classical b2-a2 linkage. The findings in this patient may be explained by an unusual genomic breakpoint downstream of the BCR gene associated with long range splicing that excluded all of the 3' BCR exons. Of the five patients with Ph-negative M-BCR non-rearranged CML studied by PFGE for BCR gene rearrangement, none had evidence of rearranged bands. We conclude that PFGE is a valuable adjunct to standard molecular techniques for the study of atypical cases of CML. Occasional patients with Ph-positive CML have breakpoints outside M-BCR. The BCR gene is probably not involved in patients with Ph-negative, M-BCR non-rearranged CML.

Characterization of human 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase gene and its expression in mammalian cells

Three β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) catalyze the oxidative conversion of Δ5-3β-hydroxysteroids to the Δ4-3-keto configuration and is therefore essential for the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Using human 3β-HSD cDNA as probe, a human 3β-HSD gene was isolated from a λ-EMBL3 library of leucocyte genomic DNA. A fragment of 3β-HSD genomic DNA was also obtained by amplification of genomic DNA using the polymerase chain reaction. The 3β-HSD gene contains a 5′-untranslated exon of 53 base pairs (bp) and three successive translated exons of 232, 165, and 1218 bp, respectively, separated by introns of 129, 3883, and 2162 bp. The transcription start site is situated 267 nucleotides upstream from the ATG initiating codon. DNA sequence analysis of the 5′-flanking region reveals the existence of a putative TATA box (ATAAA) situated 28 nucleotides upstream from the transcription start site while a putative CAAT binding sequence is located 57 nucleotides upstream from the TATA box. Expression of a cDNA insert containing the coding region of 3β-HSD in nonsteroidogenic cells shows that the gene encodes a single 42-kDa protein containing both 3β-hydroxysteroid dehydrogenase and Δ5-Δ4-isomerase activities. Moreover, all natural steroid substrates tested are transformed with comparable efficiency by the enzyme. In addition to its importance for studies of the regulation of expression of 3β-HSD in gonadal as well as peripheral tissues, knowledge of the structure of the human 3β-HSD gene should permit investigation of the molecular defects responsible for 3β-HSD deficiency, the second most common cause of adrenal hyperplasia in children.