Mapping of the dopa decarboxylase gene to the 11A band of the murine genome.

A human DOPA decarboxylase (DDC) cDNA probe of 747 base pairs has been used to map the DDC gene by in situ hybridization on mouse metaphase chromosomes. This result indicates that the gene is located on band 11A, near the erythroblastosis oncogene B (erb b) locus. This provides evidence for a synteny group on mouse chromosome 11 and human chromosome 7.     

Transforming DNA integrates into the host chromosome

A series of rat liver cotransformed cell lines have been constructed containing from 5 to 100 copies of a variant human growth hormone gene. We have used hybridization in situ to demonstrate that most, if not all, cotransformed sequences reside in a chromosome of the host cell. In each of four cell lines examined, hybridization was restricted to a single chromosomal site with no extrachromosomal sites apparent. The site was invariant within each line; however, each line revealed a different site of integration for transforming sequences. In two of the four lines, transforming DNA resided at or near the site of gross chromosomal rearrangements, in one line near an rDNA site, and in one line in the middle of an apparently normal chromosome. Thus, insertion is not restricted to a unique chromosome or chromosomal region.     

3-Aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione for circumvention of anticancer drug resistance

A series of 3-aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione was synthesized by Mannich reaction or by the transamination of 3-dimethylaminomethyl 4,11-dihydroxy- or 4,11-dimethoxynaphtho[2,3-f]indole-5,10-dione. The potency of novel derivatives was tested on a National Cancer Institute panel of 60 human tumor cell lines as well as in cells with genetically defined determinants of cytotoxic drug resistance, P-glycoprotein (Pgp) expression, and p53 inactivation. Mannich derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione with an additional amino function in their side chain, demonstrated equal cytotoxicity against the parental K562 leukemia cells and their Pgp-positive subline, whereas the latter showed approximately 7-fold resistance to adriamycin, a Pgp transported drug. 3-(1-Piperazinyl)methyl and 3-(quinuclidin-3-yl)aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione killed HCT116 colon carcinoma cells (carrying wild type p53) and their p53-null variant within the similar range of concentrations. We conclude that Mannich modification of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione, especially when cyclic diamine (e.g., piperazine, quinuclidine) is used, confers an important feature to the resulting compounds, namely, the potency for tumor cells otherwise resistant to a variety of anticancer drugs.     

Cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity.

The enzyme 5-formyl tetrahydrofolate cyclodehydrase plays an important role in the conversion of 5-formyl tetrahydrofolate to 5,10-methenyl tetrahydrofolate. A second enzyme, cyclohydrolase, converts 5,10-methenyl tetrahydrofolate to 10-formyl tetrahydrofolate. These folate derivatives play a significant part in the biosynthesis of purines. A method has been devised for the cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity which uses 5-formyl tetrahydrofolate or 5,10-methenyl tetrahydrofolate as substrate respectively, blocking possible interferences by other enzymes, and allows the nonenzymatic reduction of nitro-blue tetrazolium by 5,10-methenyl tetrahydrofolate formed by the action of the cyclodehydrase on the substrate 5-formyl tetrahydrofolate, and by 10-formyl tetrahydrofolate formed by the action of cyclohydrolase on the substrate 5,10-methenyl tetrahydrofolate, thus revealing intracellular sites of enzyme activity. The methods appear to show only intracellular localization of the blue formazan deposits of reduced tetrazolium. The distribution of positivity in cells of human blood and bone marrow is described.     

High-performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver.

The folate coenzyme 5,10-methylenetetrahydrofolate is an important folate metabolite which cannot be determined directly by HPLC near neutral pH because it dissociates to formaldehyde and tetrahydrofolate. A method for the determination of 5,10-methylenetetrahydrofolate in liver is described. This method involves (1) determination of liver 5-methyltetrahydrofolate; (2) chemical reduction of liver 5,10-methylenetetrahydrofolate (stabilized at pH 10) to 5-methyltetrahydrofolate; and (3) determination of total liver 5-methyltetrahydrofolate. Subtraction of (1) from (3) gives the concentration of 5,10-methylenetetrahydrofolate in liver.     

Novel tRNA gene organization in the 16S-23S intergenic spacer of the Streptococcus pneumoniae rRNA gene cluster.

Isoleucine and alanine tRNAs are encoded tandemly within the 16S-23S intergenic spacer of some eubacterial rRNA gene clusters. Southern hybridization analysis and DNA sequence analysis demonstrated a novel gene organization for an rRNA gene cluster on the Streptococcus pneumoniae chromosome. A sequence specifying an alanine tRNA was found within the intergenic spacer, but no sequence specifying an isoleucine tRNA was found there. Southern hybridization analysis indicated that the location of the isoleucine tRNA gene was near the 5S rRNA gene in two of four rRNA gene clusters.     

Localization of tandemly repeated DMA sequences in Arabidopsis thaliana

In-situ hybridization to interphase nuclei and chromosomes of Arabidopsis thaliana (2n= 10) shows that there are four sites of rDNA in a diploid nucleus. The sites are located on chromosomes 2 and 4, and the strength of hybridization indicates that copy number is similar at both pairs of sites. Hybridization to trisomic line 4 revealed five hybridization sites. Silver staining of nucleoli demonstrates that all four loci can be active in diploid interphase nuclei. The tandemly repeated probe pAL1 hybridizes near to the centromeres of all five chromosome pairs. In diploid interphase nuclei, 10 sites of hybridization are detected, while 15 are seen in triploid nuclei. The sites of hybridization co-localize with the centromeric heterochromatin visualized by staining DNA with the fluorochrome DAPI. The results demonstrate that molecular cytogenetics can be applied to A. thaliana and high resolution physical chromosome maps can be generated. Both probes may be useful for interphase cytogenetics, where they enable chromosome number and aneuploidy to be examined in tissues without divisions. The physical localization of these hybridization sites provides a starting point for linking RFLP and physical chromosome maps.     

Isolation of a sequence which maps close to the human sex determining gene.

A sequence mapping close to the human sex determining gene (TDF) has been isolated from a lambda library constructed with DNA derived from a chromosome transfectant hybrid cell line. This sequence is shown to be present in the DNA of X-Y interchange males at a very high frequency and, based on these studies, it is categorised with the sequence defined by the probe, GMGY3, as the closest known Y chromosome derived marker to TDF. In contrast to GMGY3, however, this locus shares no homology with any other human chromosome. Southern blot analysis also reveals specific hybridization to the Y chromosome of other primates. It therefore defines, for the first time, a conserved and Y chromosome unique locus that is near to TDF.     

Sex reversal in the mouse (Mus musculus) is caused by a recurrent nonreciprocal crossover involving the X and an aberrant Y chromosome

Satellite DNA (Bkm) from the W sex-determining chromosome of snakes, which is related to sequences on the mouse Y chromosome, has been used to analyze the DNA and chromosomes of sex-reversed (Sxr) XXSxr male mice. Such mice exhibit a male-specific Southern blot Bkm hybridization pattern, consistent with the presence of Y-chromosome DNA. In situ hybridization of Bkm to chromosomes of XXSxr mice shows an aberrant concentration of related sequences on the distal terminus of a large mouse chromosome. The XYSxr carrier male, however, shows a pair of small chromosomes, which are presumed to be aberrant Y derivatives. Meiosis in the XYSxr mouse involves transfer of chromatin rich in Bkm-related DNA from the Y-Y1 complex to the X distal terminus. We suggest that this event is responsible for the transmission of the Sxr trait.     

Chromosomal localization of three endogenous retrovirus loci associated with virus production in White Leghorn chickens.

Proposed mechanisms for the generation of endogenous retrovirus loci have been examined by determining the chromosomal distribution of these loci by means of in situ hybridization. Unlike the clustering on chromosome 1 of five endogenous retrovirus loci associated with the gs- chf- phenotype A. Tereba and S. M. Astrin, submitted for publication), three loci associated with endogenous retrovirus production (V+ phenotype) were located on three separate chromosomes. ev2, which codes for the prototype endogenous RAV-0 genome in line 7(2) chickens, was localized near the middle of the long arm of chromosome 2, ev7, coding for a noninfectious, inducible genome present in line 15B chickens, was located near the end of the long arm of the Z chromosome. A third V+ locus, designated ev14, was detected near the middle of chromosome 3. This arrangement of V+ loci is consistent with an integration mechanism employing randomly distributed integration sites in the chicken genome. In addition, these data provide evidence suggesting that the gs- chf- -associated loci may have been generated by a different mechanism.     

Human tyrosinase gene, mapped to chromosome 11 (q14----q21), defines second region of homology with mouse chromosome 7

The enzyme tyrosinase (monophenol,L-dopa:oxygen oxidoreductase; EC 1.14.18.1) catalyzes the first two steps in the conversion of tyrosine to melanin, the major pigment found in melanocytes. Some forms of oculocutaneous albinism, characterized by the absence of melanin in skin and eyes and by a deficiency of tyrosinase activity, may result from mutations in the tyrosinase structural gene. A recently isolated human tyrosinase cDNA was used to map the human tyrosinase locus (TYR) to chromosome 11, region q14----q21, by Southern blot analysis of somatic cell hybrid DNA and by in situ chromosomal hybridization. A second site of tyrosinase-related sequences was detected on the short arm of chromosome 11 near the centromere (p11.2----cen). Furthermore, we have confirmed the localization of the tyrosinase gene in the mouse at or near the c locus on chromosome 7. Comparison of the genetic maps of human chromosome 11 and mouse chromosome 7 leads to hypotheses regarding the evolution of human chromosome 11.     

Synthesis and structure-activity relationship studies of 4,11-diaminonaphtho[2,3-f]indole-5,10-diones

We describe the synthesis of derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione and their cytotoxicity for human tumor cells that express major determinants of altered anticancer drug response, the efflux pump P-glycoprotein, and non-functional p53. Nucleophilic substitution of methoxy groups in 4,11-dimethoxynaphtho[2,3-f]indole-5,10-dione with various ethylenediamines yielded the derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione, the indole containing analogues of the antitumor agent ametantrone. The cytotoxicity of novel compounds for multidrug resistant, P-glycoprotein-expressing tumor cells is highly dependent on the N-substituent at the terminal amino group of the ethylenediamine moiety. Whereas p53 null colon carcinoma cells were less sensitive to the reference drug doxorubicin than their counterparts with wild type p53, the majority of novel naphthoindole derivatives were equally potent for both cell lines, regardless of the p53 status.     

The genes for interleukins 3 and 5 map to the same locus on mouse chromosome 11

The cytokines, IL-3, IL-4, IL-5, and GM-CSF (encoded by murine genes Il-3, Il-4, Il-5, and Csfgm) belong to a family of secreted glycoprotein hormones that regulate the haemopoietic and immune systems. We demonstrate here using in situ hybridization that Il-3 and Il-5 are both probably located in the segment comprising band A5 and the proximal half of band B1 on mouse chromosome 11 with a possible location point in band B1 near its proximal interface with band A5. In studies reported elsewhere we have shown close physical linkage between Il-3 and Csfgm and also between Il-4 and Il-5. The in situ hybridization results therefore indicate that all four cytokine genes are clustered on chromosome 11 raising the possibility that they arose by ancient gene duplication.     

Centromeric repetitive DNA sequences in the genus Brassica

Representatives of two major repetitive DNA sequence families from the diploid Brassica species B. campestris and B. oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization. Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs. Such chromosome specificity is unusual for plant paracentromeric repetitive DNA. Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes. In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species.     

Localization of viral transforming sequences within marker chromosomes associated with tumor formation and progression in a murine fibrosarcoma

The low-metastatic RSV-transformed fibrosarcoma line B77-3T3 and its metastatic variant AA12, selected in vitro, have been analysed by blot and in situ hybridization with v-src and murine c-myc specific probes in order to detect molecular rearrangements underlying the transition from the low-metastatic to the high-metastatic phenotype. Previous cytogenetic analysis had evidenced that a marker chromosome of the parental tumor line (chromosome A) is replaced in the metastatic counterpart by a new marker chromosome (chromosome B), possibly arisen by duplication of a chromosome A segment, included between two C-positive regions (L. Doneda et al., 1985). In situ hybridization on chromosome spreads of the two related lines with a ³H-labelled v-src probe showed that src sequences are located within the marker chromosomes A and B, and the percentage of grains over the AA12 marker chromosome is always double that found on the B77-3T3 marker. These signals were considered to identify v-src sequences as they were found to be slightly amplified in the metastatic variant DNA by blot hybridization with the v-src probe. As regards the intrachromosomal location of the signals, most grains were clustered near the heterochromatic bands, suggesting a possible role for heterochromatic sites in tumor formation and evolution. No involvement of the A and B marker chromosomes was shown by in situ hybridization experiments with a c-myc probe. However the dosage of c-myc sequences was also found to be slightly increased in the metastatic variant DNA.     

Identification of a homeologous chromosome pair by in situ DNA hybridization to ribosomal RNA loci in meiotic chromosomes of cotton (Gossypium hirsutum).

In situ DNA hybridization with 18S-28S and 5S ribosomal DNA probes was used to map 18S-28S nucleolar organizers and tandem 5S repeats to meiotic chromosomes of cotton (Gossypium hirsutum L.). Mapping was performed by correlating hybridization sites to particular positions in translocation quadrivalents. Arm assignment required translocation quadrivalents with at least one interstitial chiasma and sufficient distance between the hybridization site and the centromere. We had previously localized a major 18S-28S site to the short arm of chromosome 9; here we mapped two additional major 18S-28S sites to the short arm of chromosome 16 and the left arm of chromosome 23. We also identified and mapped a minor 18S-28S site to the short arm of chromosome 7. Two 5S sites of unequal size were identified, the larger one near the centromere of chromosome 9 and the smaller one near the centromere of chromosome 23. Synteny of 5S and 18S-28S sites indicated homeology of chromosomes 9 and 23, while positions of the other two 18S-28S sites supplement genetic evidence that chromosomes 7 and 16 are homeologous.     

Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization.

A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.     

Assignment of the rabbit genes for alpha (PHKA) and beta (PHKB) phosphorylase kinase subunits.

The chromosomal locations of the rabbit genes for the alpha and beta subunits of phosphorylase kinase (PHKA and PHKB) were determined by in situ hybridization using rabbit cDNA probes. Our results localize PHKA to the X chromosome at the proximal end of the long arm, near the centromere, and PHKB to the same location on chromosome 5. These assignments support previously reported homoeologies of rabbit and human chromosomes.     

Assignment of the porcine calcium release channel gene, a candidate for the malignant hyperthermia locus, to the 6p11----q21 segment of chromosome 6

Several studies point to the possibility that malignant hyperthermia (MH) in pigs is caused by a defect in the calcium release channel (CRC) of skeletal muscle sarcoplasmic reticulum. The locus for MH is closely linked to the glucosephosphate isomerase (GPI) locus, near the centromere of chromosome 6. We demonstrate synteny of the genes for CRC and GPI using somatic cell hybrid lines, and assign the CRC gene to chromosome 6p11----q21 by in situ hybridization.