Chromosomal mapping of chicken mega-telomere arrays to GGA9, 16, 28 and W using a cytogenomic approach.

Four mega-telomere loci were mapped to chicken chromosomes 9, 16, 28, and the W sex chromosome by dual-color fluorescence in situ hybridization using a telomeric sequence probe and BAC clones previously assigned to chicken chromosomes. The in-common features of the mega-telomere chromosomes are that microchromosomes are involved rather than macrochromosomes; in three cases (9, 16, 28) acrocentrics are involved with the mega-telomeres mapping to the p arms. Three of the four chromosomes (9, 16, W) encode tandem repeats which in two cases (9 and 16) involve the ribosomal DNA arrays (the 5S and 18S-5.8S-28S gene repeats, respectively). All involved chromosomes have a typical-sized telomere on the opposite terminus. Intra- and interindividual variation for mega-telomere distribution are discussed in terms of karyotype abnormalities and the potential for mitotic instability of some telomeres. The diversity and distribution of telomere array quantity in the chicken genome should be useful in contributing to research related to telomere length regulation - how and by what mechanism genomes and individual chromosomes establish and maintain distinct sets of telomere array sizes, as well as for future studies related to stability of the chicken genome affecting development, growth, cellular lifespan and disease. An additional impact of this study includes the listing of BAC clones (26 autosomal and six W BACs tested) that were cytogenetically verified; this set of BACs provide a useful tool for future cytogenetic analyses of the microchromosomes.

Telomeres in the chicken: genome stability and chromosome ends.

Telomeres are the complex nucleoprotein structures at the termini of linear chromosomes. Telomeric DNA consists of a highly conserved hexanucleotide arranged in tandem repeats. Telomerase, a ribonucleoprotein of the reverse transcriptase family, specifies the sequence of telomeric DNA and maintains telomere array length. Numerous studies in model organisms established the significance of telomere structure and function in regulating genome stability, cellular aging, and oncogenesis. Our overall research objectives are to understand the organization of the telomere arrays in chicken in the context of the unusual organization and specialized features of this higher vertebrate genome (which include a compact genome, numerous microchromosomes, and high recombination rate) and to elucidate the role telomeres play in genome stability impacting cell function and life span. Recent studies found that the chicken genome contains three overlapping size classes of telomere arrays that differ in location and age-related stability: Class I 0.5 to 10 kb, Class II 10 to 40 kb, and Class III 40 kb to 2 Mb. Some notable features of chicken telomere biology are that the chicken genome contains ten times more telomeric DNA than the human genome and the Class III telomere arrays are the largest described for any vertebrate species. In vivo, chicken telomeres (Class II) shorten in an age-related fashion and telomerase activity is high in early stage embryos and developing organs but down-regulates during late embryogenesis or postnatally in most somatic tissues. In vitro, chicken cells down-regulate telomerase activity unless transformed. Knowledge of chicken telomere biology contributes information relevant to present and future biotechnology applications of chickens in vivo and chicken cells in vitro.

The chicken telomerase RNA gene: conservation of sequence, regulatory elements and synteny among viral, avian and mammalian genomes.

Telomerase RNA (TR) is essential for telomerase activity and the maintenance of telomere length in proliferating cell populations. The objective of the present research was to define the cytogenetic and molecular genomic organization of chicken TR (chTR). The chTR exists as a single copy gene (TERC, alias TR), mapping to chromosome 9 (GGA9). The loci on the q arm of GGA9 map to three chromosomes in human with five of the nine GGA9q loci mapping to HSA3q. Sequencing of the chTERC locus (3,763 bp) from the UCD 001 genome (Red Jungle Fowl) included: 604 bp 5', 465 coding, and 2,694 bp 3' (from -604 to +3159). Sequence analysis included homology searches conducted on several levels including comparisons among different chicken genotypes, Marek's disease virus (MDV) sequences, plus human and murine. We provide evidence for distal 5' and 3' sequence homology between chTERC and the MDV genome among other known regions of homology (promoter and coding), elaborate on 5' transcription factor binding motifs among the various genomes as well as show type and number of TERT-related motifs 3' of chicken TR (e.g., Sp1, c-Myb, c-Myc, AP2, among others). Surrounding the gene are more than 25 Sp1 sites, over 20 oncogene transcription factor binding motifs and numerous hormonal and other specialized binding motifs. Knowledge of 5' and 3' chTERC regulatory elements will be useful for investigating normal control mechanisms during growth and development as well as investigating the potential for dysregulation of this important gene during oncogenesis, especially among different genotypes.

Correlation of structure and triboluminescence for tetrahedral manganese(II) compounds.

To investigate the structural basis of triboluminescence, several known tetrahedrally coordinated Mn(II) complexes have been synthesized according to literature methods and their crystal structures have been determined by X-ray diffraction. Among them, (MePh(3)P)(2)[MnCl(4)] (2), a = 15.4804(4) A, cubic, space group P2(1)3, Z = 4; (Et(4)N)(2)[MnBr(4)] (4), a = 13.362(1) A, c = 14.411(1) A, tetragonal, space group P42(1)m, Z = 4; MnBr(2)(OPPh(3))(2) (7), a = 9.974(1) A, b = 10.191(3) A, c = 10.538(2) A, alpha = 65.32(1) degrees, beta = 63.49(1) degrees, gamma = 89.44(2) degrees, triclinic, space group P1, Z = 1; and MnBr(2)(OAsPh(3))(2) (10), a = 17.816(3) A, b = 10.164(1) A, c = 18.807(3) A, orthorhombic, space group Pca2(1), Z = 4 were reported to be triboluminescent and (Me(4)N)(2)[MnCl(4)] (3), a = 9.016(3) A, b = 36.90(2) A, c = 15.495(3) A, beta = 90.72(3) degrees, monoclinic, space group P2(1)/n, Z = 12, and MnI(2)(OAsPh(3))(2) (11), a = 10.094(4) A, b = 10.439(2) A, c = 34.852(2) A, alpha = 83.17(4) degrees, beta = 86.09(2) degrees, gamma = 75.16(3) degrees, triclinic, space group P1, Z = 4, were reported to be not triboluminescent. The result supports the correlation between space group acentricity and triboluminescence activity.

Di- and trinuclear complexes with the mono- and dianion of 2,6-bis(phenylamino)pyridine: high-field displacement of chemical shifts due to the magnetic anisotropy of quadruple bonds.

The monoanion of 2,6-bis(phenylamino)pyridine (HBPAP(-)) has been found to support quadruply bonded Cr(2)(4+) and Mo(2)(4+) units in Cr(2)(HBPAP)(4) (1) and Mo(2)(HBPAP)(4) (2). The corresponding dianion BPAP(2)(-) was able to stabilize the trinuclear complexes, (TBA)(2)Cr(3)(BPAP)(4) (3) and (TBA)(2)Ni(3)(BPAP)(4) (4), where TBA is the tetrabutylammonium cation. The dinuclear complexes have the typical paddlewheel configuration with Cr-Cr distances of about 1.87 A and a Mo-Mo distance of 2.0813(5) A and exhibit a high-field displacement of the corresponding N-H signals caused by the magnetic anisotropy of the quadruple bonds. For the trinuclear complexes, 3 has a linear chain of three chromium atoms arranged in an unsymmetrical fashion with two chromium atoms paired to give a quadruply bonded unit (Cr-Cr distance: 1.904(3) A) and an isolated, square planar Cr(II) unit at 2.589(3) A from the dimetal unit. On the other hand, the three nickel atoms in 4 are evenly spaced, having Ni.Ni distances of 2.3682(8) A. The trinuclear compounds show a twisted conformation with an overall torsion angle of about 30 degrees.

Tuning the metal-metal bonds in the linear tricobalt compound Co3(dpa)(4)Cl2: bond-stretch and spin-state isomers.

Sixteen crystal structures have been determined for the Co3(dpa)(4)Cl2 (1) molecule in the following five crystalline solvates: 1.0.85(C2H5)(2)O.0.15CH2Cl2 (at 120, 213, 296 K); 1.C(4)H(8)O (at 120, 295 K); 1.C(6)H(6) (at 170, 213, 260, 316 K); 1.C(6)H(12) (at 120, 213, 295 K); and 1.1.75C(7)H(8).0.5C(6)H(14) (at 90, 110, 170, 298 K). For 1.0.85(C(2)H(5))(2)O.0.15CH2Cl2 the molecule of 1 is almost symmetrical at 120 K (Co-Co distances of 2.3191(3) and 2.3304(3) A) and remains so at 296 K (2.2320(3) and 2.3667(4) A). For 1.C(4)H(8)O the Co(3) chain is precisely symmetric at both 120 and 295 K though the Co-Co distances increase from 2.3111(4) to 2.3484(4) A as the temperature rises. Compound 1.C(6)H(6) is isomorphous with 1.C(4)H(8)O at 213 and 295 K and has rigorously symmetrical molecules at these two temperatures. Between 213 and 120 K the space group changes from Pccn to P2(1)/c, so that a symmetrical arrangement is no longer required and the two Co-Co distances then differ slightly (by 0.013 A). For 1.C(6)H(6) there is a phase change between 316 K (Pca2(1)) and 260 K (Pna2(1)). At all four temperatures, however, the molecule is almost symmetrical, with the two independent Co-Co distances never differing by more than 0.026 A. 1.1.75C(7)H(8).0.5C(6)H(14) contains, at all temperatures between 90 and 298 K, two crystallographically independent molecules, each of which is distinctly unsymmetrical at 298 K (Co-Co distances of 2.312(2) and 2.442(2) A for one and 2.310(2) and 2.471(2) for the other). In the first of these the distances converge to a much smaller separation (0.056 A) at 90 K while in the second the difference decreases to only 0.006 A at 90 K. Magnetic susceptibility measurements from 1.8 to 350 K indicate in each case that a gradual spin crossover, from a doublet to a quartet state, occurs over this temperature range.

Linear trichromium complexes with the anion of 2,6-di(phenylimino)piperidine.

The anion of 2,6-di(phenylimino)piperidine (DPhIP) has been found to support linear chains of three metal atoms. Three new compounds, [Cr3(DPhIP)4Cl]Cl.(1).5CH2Cl2.0.5H2O (1.1.5CH2Cl2.0.5H2O), [Cr3(DPhIP)4(CH3CN)]- (PF6)2.H2O.4CH3CN (2.H2O.4CH3CN), and [Cr3(DPhIP)4(F)(CH3CN)](BF4)2.5CH3CN (3.5CH3CN), have been synthesized and characterized by X-ray crystallography. Compound 1 has a linear chain of three chromium atoms arranged in an unsymmetrical fashion, with two of them forming a quadruply bonded unit (Cr-Cr distance 1.932(2) A) and the third being a non-metal-metal-bound 5-coordinate unit (Cr...Cr distance 2.659(2) A). The fifth coordination site is occupied by a chloride ion, and another chloride ion is located in the interstices of the crystal. The trimetal unit in compound 2 is structurally similar to that in compound 1 except that the axial ligand in 2 is a CH3CN molecule. Compound 3 is an oxidation product prepared by reaction of 1 with AgBF4. Here, a square pyramidal CrIII unit, FCrN4, and a Cr-Cr quadruply bonded (Cr-Cr distance 1.968(2) A) unit, with an axially coordinated acetonitrile molecule, form the trichromium chain. The CrIII...CrII separation of 2.594(2) A in 3 is too long to be considered a bonding interaction.

Completion of the series of M2(hpp)4Cl2 compounds from W to Pt: the W, Os, and Pt compounds.

The series of M2(hpp)4Cl2 complexes (hpp is the anion of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine) from M = W to M = Pt has been completed by the preparation and characterization of those with M = W, Os, and Pt. W(hpp)4Cl2 (1) has a W-W distance of 2.250(2) A, is diamagnetic, and can be assigned a W-W triple bond based on a sigma 2 pi 4 electron configuration. Os2(hpp)4Cl2 (2) has an Os-Os distance of 2.379(2) A and displays a temperature-independent paramagnetism. It can be assigned a sigma 2 pi 4 delta 2 delta*2 configuration. Pt2(hpp)4Cl2 has a Pt-Pt distance of 2.440(1) A and is diamagnetic. A bond order of 1, based on a configuration in which only the sigma* orbital is empty, is consistent with these data.

Linear trichromium complexes with direct Cr to Cr contacts. 1. Compounds with Cr3(dipyridylamide)4(2+) cores.

The preparation and structures of seven compounds that contain the Cr3(dpa)4(2+) core (dpa = the anion di(2-pyridyl)amide) are reported. The magnetic properties of several have been measured. In each case there are anionic ligands at each end of the Cr3(6+) chain, sometimes identical (2Cl-, 2CCPh-), sometimes different (Cl-, BF4-; Cl-, PF6-). Several of these compounds have a symmetrical arrangement of the three Cr atoms, with the two Cr-Cr distances equal at ca. 2.36 A, while others have an unsymmetrical arrangement. In the most extreme case the two Cr-Cr distances are 2.00 and 2.64 A. The electronic structures and the remarkable flexibility of the Cr3 arrangement are discussed.

Linear trichromium complexes with direct Cr to Cr contacts. 2. Compounds with Cr3(dipyridylamide)4(3+) cores.

Seven compounds having in common a Cr3(dpa)4(3+) core (dpa = di(2-pyridyl)amide ion) have been prepared and all shown to have an unsymmetrical chain of three Cr atoms. This chain can be described as a pair of quadruply bonded Cr(II) atoms to which a Cr(III) atom is attached. No symmetrical chain has been found, contrary to a previous preliminary report. The seven compounds have been well characterized crystallographically, and their short and long Cr to Cr distances (A, in parentheses) are: 1 [Cr3(dpa)4Cl2]Cl.2CH2Cl2.THF (2.12, 2.47), 2 [Cr3(dpa)4Cl2]AlCl4.CH2Cl2 (2.011, 2.555), 3 [Cr3(dpa)4Cl2]FeCl4.CH2Cl2 (2.009, 2.562), 4 [Cr3(dpa)4Cl2]I3.THF.2H2O (2.08, 2.49), 5 [Cr3(dpa)4Cl2]PF6.2CH2Cl2 (2.08, 2.48), 6 [Cr3(dpa)4(BF4)F]BF4.2CH2Cl2 (1.900, 2.595), 7 [Cr3(dpa)4ClF]BF4.CH2Cl2.C6H14 (2.039, 2.507). Magnetic susceptibility measurements on 1 and 2 reveal mueff = 3.85 +/- 0.05 muB from 10 to 300 K.

Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice.

There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.

Organ distribution of experimental metastases of a human colorectal carcinoma injected in nude mice.

The metastatic behavior of the HT-29 human colorectal carcinoma cell line was studied following injection into nude mice by different routes. After intrasplenic injection, experimental metastases formed in the livers of most mice. Variant lines were established in culture from the liver lesions and from tumors growing at the site of injection, the spleen. Cells of the HT-29 LMM line exhibited slightly enhanced ability to form liver metastases compared with cells of the non-selected parent line. When injected i.v., the HT-29 cells produced only a few small experimental metastases in the lungs, but in most of the mice macroscopic tumors were found in various lymph nodes and the interscapular fat. Analyses of the distribution of IdUrd-labeled cells did not reveal a preferential localization of the HT-29 cells in sites where metastases subsequently formed. This suggested that the growth of the human colon carcinoma cells in those sites might be the result of a stimulatory interaction between the tumor and host cells as opposed to growth in sites such as the lungs, where numerous cells arrested after i.v. injection but only a few, small metastases were seen 60 days later.