Faculty Bibliography

We have mapped the human gene for ferrochelatase (FECH; ferroheme-protolyase, EC 4.99.1.1) to chromosome 18 by hybridization of cDNA to sorted chromosomes. The probe was obtained by PCR-directed amplification of a human marrow cDNA library in lambda gt 10. Subchromosomal localization of ferrochelatase to 18q22 was determined by chromosomal hybridization in situ using a human ferrochelatase genomic clone in lambda EMBL 3 that contained a 20-kb insert. Since ferrochelatase activity is deficient in patients with the inherited disease erythropoietic protoporphyria, a locus for this disease may be assigned to 18q22, one of few monogenic defects that have been mapped to this chromosome.

We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.

A method for mapping nonisotopically labeled probes to human metaphase chromosomes that can be used with laser scanning confocal microscopy has been developed. Only a limited number of wavelengths are available from the argon ion lasers used in most commercial instruments and therefore a method that allowed the visualization of bands on human chromosomes stained with propidium iodide and, simultaneously, the detection of hybridization signals using FITC-labeled antibodies was developed. The confocal microscope was used to map single-copy probes to chromosome bands and the positions of the probes on the R-banded chromosomes corresponded to map positions previously determined on Hoechst 33258-stained chromosomes (G-banded). A comparison of confocal imaging of single-copy hybridization signals with conventional fluorescence microscopy and high-sensitivity video cameras revealed little difference in sensitivity but greater resolution of chromosome bands with the confocal microscope. The polymerase chain reaction was used to prepare nonisotopically labeled probes for in situ hybridization and to amplify Alu and KpnI family repeats from cloned DNA to be used to suppress hybridization of these repeat sequences so that a cosmid probe could be mapped to a chromosome band.

Five polymorphic DNA segments from human chromosome 3, that are frequently deleted in lung carcinoma were mapped by non-isotopic in situ hybridization to metaphase chromosomes. The DNA segment D3S3 mapped to 3p13-p14.2, D3S6 to 3p14.3-p14.5, D3S48 to distal 3p21-p22, ERBA beta to 3p24.3 and ERBA2 to 3p24.3. The map location of ERBA beta and ERBA2 was confirmed by re-mapping each probe in combination with D3S6 as a marker for 3p14.

DNA was prepared from tumour and normal tissue from 48 patients representing all common histological types of nonsmall-cell lung cancer. Using eight DNA probes, which detect nine restriction enzyme fragment length polymorphisms (RFLP) on chromosome 3, we established that among the 44 informative patients 32 had lost alleles on the short arm of one of their copies of chromosome 3. Of these 32, at least 13 had also lost alleles on the long arm of chromosome 3, suggesting that the whole chromosome might be lost. For one patient, cytogenetic analysis indicated that the mechanism of allelic loss was reciprocal translocation followed by chromosomal loss of one of the reciprocal products. Two patients with allelic loss distal to the D3S3 locus (which maps to 3p13-14) retained heterozygosity at that locus. These results indicate that loss of alleles on the short arm of chromosome 3 is a common event in lung tumours of the nonsmall-cell type, that this loss occurs by a variety of chromosomal mechanisms, and that the minimally deleted region is 3p13-14----3pter.

In the nematode Caenorhabditis elegans, microtubules with 15 protofilaments are a specialized feature of six touch-receptor neurons; microtubules found in other C. elegans neurons have 11 protofilaments. Mutations in the gene mec-7 result in touch-insensitive animals whose touch cells lack the 15-protofilament microtubules. We have characterized 54 mutations in the mec-7 gene. The absence of mec-7 activity results selectively in the recessive loss of touch sensitivity. Partial loss-of-function alleles result in a partial loss of touch sensitivity. Dominant mutations, which are isolated at an unusually high proportion, may encode abnormal products. We have cloned the mec-7 gene; it encodes a beta-tubulin which is 90-93% identical to vertebrate beta-tubulin. Our results are consistent with the hypothesis that tubulin heterogeneity contributes to the formation of structurally and functionally distinct sets of microtubules.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDHase) is encoded by four genes designated gpd-1 through gpd-4 in the nematode Caenorhabditis elegans. gpd-1 has been isolated and sequenced, and is shown here to have a nearly identical copy (gpd-4) with respect to coding and regulatory flanking sequence information as well as to the placement of its two introns. Both genes, which are separated by 250,000 to 300,000 base-pairs were assigned to chromosome II by in situ hybridization and physically linked to a DNA polymorphism located near unc-4 on the genetic map. The genes gpd-2 and gpd-3 are also nearly identical with each other but differ from the gpd-1 and gpd-4 pair with respect to the positions of their two introns and a cluster of amino acid changes within the amino-terminal region of the enzyme. Furthermore, one gene from each pair (gpd-4 and gpd-2) exhibits a single amino acid substitution at positions heretofore known to be conserved in all other systems so far examined including the extreme thermophiles. gpd-2 and gpd-3 are organized as a direct tandem repeat separated by only 244 base-pairs. They have been assigned to an 85,200 base-pair contig that maps to the left end of the X chromosome. The absence of gpd-3 from C. elegans var. Bergerac was used as a marker to map the gpd-2,3 gene pair near unc-20. Northern analyses have shown that gpd-1 and gpd-4 are preferentially expressed in embryos, while the expression of gpd-2 and gpd-3 increases during postembryonic development. These analyses indicate that the gpd-1,4 gene pair encodes the minor isoenzyme, GAPDHase-1, present in all cells of the nematode while the other gene pair (gpd-2,3) encodes the major isoenzyme, GAPDHase-2, preferentially expressed in the bodywall muscle. The G + T-rich and T-rich regions essential for vertebrate beta-globin polyadenylation were also observed for gpd-3.

Cytogenetic abnormalities involving chromosome 14 band q32 are consistently observed in human T-cell tumors. Patients with ataxia-telangiectasia (AT) are especially prone to development of these tumors, which frequently carry either inversion inv(14)(q11;q32) or translocation t(14;14) (q11;q32) chromosomes. We have previously shown that the cytogenetic breakpoints of one t(14;14)(q11;q32) chromosome and two inv(14)(q11;q32) chromosomes in T-cell tumors from AT and non-AT patients join the T-cell receptor alpha chain locus, at chromosome band 14q11, with a region(s) at 14q32 centromeric of the immunoglobulin heavy chain variable region (VH) gene IGHV. We now show that these two inv(14) breakpoints are linked by 2.1 kb of germ-line 14q32 DNA and that the three breakpoints define, by in situ hybridization analysis, a single locus at chromosome band 14q32.1 located about 15-20 million base pairs on the centromeric side of the IGH locus. Sequence analysis of the 14q32.1 breakpoint regions indicates that abnormal recombination does not universally result from mistaken V-D-J joining (D = diversity region; J = joining region). Therefore, we invoke a tumor selection model to describe the role of the 14q32.1 locus in tumor development.