Dan Röhme

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.

Radiation fusion hybrids for human chromosomes 3 and X generated at various irradiation doses

We have used a gamma-irradiation (2.5–25 krads) cell fusion procedure to generate human-hamster somatic cell hybrids (IHB, irradiated human fragments in B14–150 cells), retaining small fragments derived from human chromosomes 3 and X. By using Alu-element mediated PCR amplification and dot-blot hybridization with human alphoid or total human DNA as probes, 86 positive hybrids were identified and selected for further analysis. Nonisotopic fluorescence in situ hybridization (FISH) with human DNA in a set of eight hybrids demonstrated the presence of from one to eight human fragments per cell independent of irradiation dose. In contrast, a significant dose-dependent variation of fragment sizes was shown in the analysis of the 86 hybrids with markers previously mapped to 3p (seven markers) and to Xq (21 markers). Using the Xq27–28 region as a model, 40% of the hybrids generated at 5 krads or less were found to have retained fragments in the range of 3–30 Mb, 10% retained the whole chromosome arm, and the remaining 50% retained fragments of less than 2–3 Mb. The proportion of fragments of 3 Mb or larger decreased rapidly at higher irradiation doses and was very low (less than 6%) in hybrids generated at 25 krads. Upon further characterization, the 86 hybrids analyzed here will provide a mapping panel for the entire chromosomes 3 and X with an estimated resolution in the range of 1–2 Mb on average, a size range amenable to PFGE and YAC contig mapping.

[37] Microcloning of mammalian metaphase chromosomes

Publisher Summary This chapter focuses on microcloning of mammalian metaphase chromosomes. Microcloning may be used to obtain libraries of clones from individually dissected mammalian chromosomes as well as chromosome regions. The numbers of chromosomes needed for linkage studies and for starts of chromosome walks are moderate, about 100–500 genome equivaleuts. Microcloning is also the method of choice when the amount of material is limited or where sufficient amounts of bulk isolated specific chromosomes cannot easily be obtained. With the currently used equipment for microdissection relatively coarse dissections can be made. For work with higher resolution it will be necessary to develop the manipulator—for example, with piezoelectric control, and television monitor. Another line to pursue is to investigate to what extent banding techniques can be combined with microcloning work. Such developments are highly desirable, among others in view of increasing needs for polymorphic DNA markers in human linkage studies.

Different pathways for chromosomal integration of transfected circular pSVneo plasmids in normal and established rodent cells.

The chromosomal integration of transfected circular plasmid pSV2neo molecules was investigated in normal and established mammalian fibroblasts, including two secondary cultures of normal mouse fibroblast lines, one established mouse-hamster hybrid cell line, R44, and the human fibrosarcoma line, HT1080. The physical organization of the integrated molecules was studied by restriction analysis. The results showed that whereas the normal fibroblasts predominantly integrated one head-to-tail partial dimeric molecule, the established cells predominantly integrated distinctly different molecular forms including deleted monomeres (HT1080) and various complex concatemeric molecules (R44), and frequently at more than one chromosomal site (R44). We also constructed a head-to-tail dimeric version of the plasmid, which in the case of the normal fibroblasts again integrated as a partial dimeric molecule in at least 50% of these cells. This result excluded the possibility that the normal mouse transfectants were selected for the integration of two functional neo genes. Thus, it is concluded that the distinctly different molecular forms integrated in normal and established cells demonstrate the operation of different integration pathways, the possible nature of which is discussed.

Ageing and the fusion sensitivity potential of human cells in culture: Relation to tissue origin, donor age, and in vitro culture level and condition

Cell fusion was induced by inactivated Sendai virus in different human diploid cell lines. These were derived from the kidney, lung or skin originating from embryos or adult donors and representing predominantly epithelioid cells (kidney) and fibroblastic cells (lung and skin). The fusion sensitivity (FS) potentials of these cell lines were determined and related to various aspects of cell ageing. In case of the fibroblastic lines, an inverse relation was demonstrated both to the culture age in vitro and to the donor age. The FS potential of embryonal fibroblasts decreased some 40-50% during the in vitro cultivation. In comparison to exponentially growing fibroblasts, the FS potentials were higher in cells in the stationary phase of growth. This was shown to correlate well with the fact that the life-span in calendar time also increased in cultures predominantly grown in the stationary phase. In the case of the kidney cells, the FS potentials were some 50% higher than those of fibroblasts. Since the cellular manifestation of the FS potential most likely primarily resides in the cell membrane-cytoskeleton structure, the results emphasize the importance of this system in relation to ageing.

On the origin of coding sequences from random open reading frames

SummaryThe size distribution of 411 randomly selected mammalian exons was investigated. This distribution was found to be unimodal with a frequency maximum of 120 bp. Detailed analysis of the distribution demonstrated that larger exons (>150 bp) have a high goodness of fit to the size distribution of open reading frames (ORFs) in a random sequence, i.e., (61/64)t in which t is the number of triplets. Based on this observation, the general character of the total exon size distribution suggested that this could be defined by a theoretical distribution by superimposing a sigmoid function on the ORF generating function, i.e., (61/64)t×fs(t)×E in which fs(t) is a sigmoid function and E is a constant. We tested this distribution for fitness to the exon distribution using two sigmoid functions. fs(t)=Φ(t) and fs(t)=Bekt/1+Bekt. In both cases a very high goodness of fit was attained. It is concluded that exons have been generated from ORFs in random sequences, that ORFs larger than 150 bp have been selected, irrespective of size, as exons, and that a lower size limit exists below which the probability of an ORF being selected as an exon is very low. These results provide evidence at the molecular level to support the ideas that (1) larger exons have been selected from random ORFs without primary correlation to structural or functional properties at the protein level, (2) there exists a restriction on smaller ORFs to be selected as exons, and (3) the interrupted coding sequences found in eukaryotes represent the ancient form of gene organization that existed prior to the divergence of prokaryotes and eukaryotes.

Monochromosomal mouse microcell hybrids containing inserted selectableneo genes

Normal mouse fibroblasts at early passage levels were used as a starting material to construct mouse-hamster microcell hybrids (MCH). Theneor gene, carried on the pSV2neo and pZIP-NeoSV(X)1 plasmids, was introduced into the mouse fibroblasts by gene transfection and retroviral infection, respectively, prior to microcell hybridization into the E36 Chinese hamster cell line. In total about 180 MCH clones were isolated and their amount of mouse DNA was estimated by dot-blot analysis. About 50% of the transfection based hybrids (T-hybrids) showed signals indicating one mouse chromosome, less than 10% more than one mouse chromosome, and the remaining clones contained only subchromosomal amounts of mouse DNA. In the infection-based hybrid series (I-hybrids) more than 95% showed only subchromosomal mouse DNA content. Chromosomal integration analysis verified the presence ofneor insertions in all 42 hybrid clones analyzed. C-banding analysis verified 14 of 15 hybrids scored as monochromosomals on dot blots. Chromosome fragmentation in T-type MCH was found to be (1) nonrandom, preferentially occurring in MCH derived from certain transfectants, (2) late in clonal establishment, and (3) essentially not related to prolonged cultivation in vitro. Once established, most T-type MCH clones including mono- and subchromosomal hybrids were essentially stable during prolonged cultivation. In contrast MCH initially containing several mouse chromosomes tend to lose the nonselectable ones during prolonged cultivation. In total we estimate the number of independent monochromosomal MCH derived in this study to more than 30.

Quantitative Cell Fusion: Derivation and Application of Theoretical Models

Cell fusion experiments were made on ten cell lines representing seven mammalian species, using inactivated Sendai virus. The extent of fusion was determined microscopically and tabulated as frequencies of cells with different numbers of nuclei. Expected distributions were derived theoretically under certain assumptions concerning the fusion process. A random model was assumed according to which the tendency to fuse depends only on the cell size, expressed as the number of nuclei present in the cell. Three distributions were derived, which were referred to as the simple, additive, and multiplicative models. The additive model pertained to fusions made in cell suspensions and the multiplicative one mainly to fusions in fibroblast monolayers.

Physical map of small cell lung cancer deletion region on short arm of human chromosome 3 (3p13-22) based on radiation fusion hybrid analysis

Deletion of DNA sequences from various regions of the short arm of human chromosome 3 (3p13–14, 3p21, and 3p25) has been observed during the development of a variety of solid tumors, including lung and renal cell carcinomas. In this study we have used a set of radiation fusion hybrids to generate a physical map of chromosome 3p to orient the search for putative tumor suppressor genes. Eighty-six human-hamster radiation fusion hybrids were screened on Southern blots for the retention of 55 human chromosome 3p DNA markers. The high marker density enabled us to identify a set of successively overlapping chromosome fragments in the 3p13–22 area guided by eight markers with previously known order. Twenty-four map intervals were suggested using breakpoints determined by partial fragment overlaps. The final order between the markers derived is consistent with previous information about localizations for 26 of the markers to three larger cytogenetic intervals.

Identification of human chromosome region 3p14.2-21.3-specific YAC clones using alu-PCR products from a radiation hybrid

Deletion of DNA sequences from at least three different regions on the short arm of human chromosome 3 (3p13–14, 3p21 and 3p25) are frequently observed during the development of many solid tumors, including lung cancers and renal cell carcinomas. In order to physically characterize the 3p21 region, we previously identified a radiation fusion hybrid that contained about 20 megabases of DNA from chromosome region 3p14.2–p21.3. In this study total Alu-PCR products from this hybrid were used as a probe to isolate 86 yeast artificial chromosomes (YAC) clones from a 620-kb average insert YAC library (ICRF). Sixty-nine Alu-PCR markers, generated from the YACs, and seven PCR primers were used to screen for overlaps between individual clones. Seven contigs were identified encompassing 32 YAC clones. Based on previous information about localization of the PCR primers, the three largest contigs could be assigned to smaller subregions between 3p14.2 and 3p21.3. By this work a large proportion of the 3p14.2–21.3 region is covered with large-insert YAC clones.