Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.     

Evidence for synergistic anion binding to iron in ovotransferrin complexes from resonance Raman and extended X-ray absorption fine structure analysis

The 2,3-dihydroxybenzoate and thioglycolate complexes of iron(III)-ovotransferrin have been studied with resonance Raman and extended x-ray absorption fine structure spectroscopies, respectively, to obtain evidence for the coordination of the synergistic anion to the iron center. The dihydroxybenzoate complex exhibits resonance-enhanced Raman vibrations arising from both the endogenous tyrosinates and the added dihydroxybenzoate. A comparison of the extended x-ray absorption fine structure spectra of the carbonate and thioglycolate complexes shows a large feature at about 1.95 A assigned to Fe-(O,N) interactions. The latter complex exhibits an added feature at 2.32 A assigned to an Fe-S interaction. These experiments demonstrate that the Lewis base functions in the synergistic anions coordinate to the iron in ovotransferrin.     

Cyanide and methylisocyanide binding to the isolated iron-molybdenum cofactor of nitrogenase

19F NMR and x-ray absorption experiments have been performed with both the isolated FeMo cofactor and the MoFe protein of nitrogenase in search of direct evidence for substrate or inhibitor binding. Using 19F NMR as a probe and p-CF3C6H4S- as the receptor ligand, the data show that the nitrogenase inhibitors CN- and CH3NC bind to the isolated FeMo cofactor-RFS- complex in N-methylformamide with a finite formation constant. Their binding increases the electronic relaxation time of the complex and increases the life-time of the FeMo cofactor-p-CF3C6H4S- bond, Parallel molybdenum K edge and extended x-ray absorption fine structure experiments show that CH3NC does not bind to molybdenum. Although CO and N3- both relieve CN- and CH3NC inhibition of electron flow through nitrogenase, unlike the latter, they do not appear to bind to isolated FeMo cofactor. In experiments with the dithionite-reduced MoFe protein, we did not detect any changes in the molybdenum K edge or extended x-ray absorption fine structure spectra upon addition of CO, N2, C2H2, NaCN, CH3NC, or azide demonstrating that either these substrates and inhibitors do not bind to molybdenum or that the FeMo cofactor site of nitrogenase is inaccessible to substrate binding except under turnover conditions.     

Identification of a new mutation in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

A mutation involving an A-to-G nucleotide replacement at position 985 of the medium-chain acyl-CoA dehydrogenase (MCAD) cDNA was found in homozygous form in 18 unrelated MCAD-deficient families and in heterozygous form in 4 families. By PCR amplification and sequencing of cDNA from a compound heterozygote, we have detected a new mutation in an MCAD-deficient patient in whom one MCAD allele produces mRNA that is missing 4 bp in the MCAD cDNA, while the other allele carries the A-to-G-985 mutation. The presence of this 4-bp deletion was confirmed in the patient's genomic DNA by dot-blot hybridization with allele-specific oligonucleotide probes and by restriction analysis of PCR products. A rapid screening test for this 4-bp deletion was developed, based on mismatched primer PCR amplification. The deletion created a new restrictive-enzyme site which yielded two DNA fragments. The 4-bp deletion was not found in the three remaining MCAD chromosomes not harboring the A-to-G-985 mutation, nor it was present in 20 chromosomes from 10 unrelated normal Caucasians. The PCR-based method for screening these two mutations can detect over 93% of all MCAD mutations.     

Linkage disequilibrium between the FES, D15S127, and BLM loci in Ashkenazi Jews with Bloom syndrome.

Bloom syndrome (BS) is more common in the Ashkenazi Jewish than in any other population. Approximately 1 in 110 Ashkenazi Jews carries blm, the BS mutation. The locus mutated in BS, BLM, maps to chromosome subband 15q26.1, tightly linked to the proto-oncogene FES. We have investigated the basis for the increased frequency of blm in the Ashkenazim by genotyping polymorphic microsatellite loci tightly linked to BLM in affected and unaffected individuals from Ashkenazi Jewish and non-Ashkenazi populations. A striking association of the C3 allele at FES with blm (delta = .422; p = 5.52 x 10(-7)) and of the 145-bp and 147-bp alleles at D15S127 with blm (delta = .392 and delta = .483, respectively; p = 2.8 x 10(-5) and p = 5.4 x 10(-7), respectively) was detected in Ashkenazi Jews with BS. This linkage disequilibrium constitutes strong support for a founder-effect hypothesis: the chromosome in the hypothetical founder who carried blm also carried the C3 allele at FES and either the 145-bp or the 147-bp allele at D15S127.