Variability of chromosome 1 with HSR insertions in natural and synanthropous populations of house mouse Mus musculus L. 1758

House mice carrying aberrant chromosome 1 with an insertion of homogeneously stained regions (HSR) have been studied. The mice were collected in the North Caucasus, Chita and Amur oblasts, Spitzbergen and Kunashir Islands, Altai krai, Khabarovsk krai, Primorye, Sakhalin, Kamchatka, Turkmenistan, and Kazakhstan. In these mice, the aberrant chromosomes were assigned to the "Asian" type, i.e. they carried two HSR insertions. The aberrant chromosome 1 in house mice from different geographic regions was shown to differ in size of HSR, staining intensity, and some other features of Q-H, C, and G-banding, which suggests independent origin of this aberration in house mouse populations from different taxa and regions. A novel variant of chromosome 1 in mice of the subspecies M. m. wagneri was found.     

Variability of chromosome 1 with HSR insertions in natural and synanthropous populations of house mouse <Emphasis Type="Italic">Mus musculus</Emphasis> L. 1758

House mice carrying aberrant chromosome 1 with an insertion of homogeneously stained regions (HSR) have been studied. The mice were collected in the North Caucasus, Chita and Amur oblasts, Spitzbergen and Kunashir Islands, Altai krai, Khabarovsk krai, Primorye, Sakhalin, Kamchatka, Turkmenistan, and Kazakhstan. In these mice, the aberrant chromosomes were assigned to the “Asian” type, i.e. they carried two HSR insertions. The aberrant chromosome 1 in house mice from different geographic regions was shown to differ in size of HSR, staining intensity, and some other features of Q-H, C, and G-banding, which suggests independent origin of this aberration in house mouse populations from different taxa and regions. A novel variant of chromosome 1 in mice of the subspecies M. m. wagneri was found.     

DNA amplification associated with double minutes originating from chromosome 19 in mouse hepatocellular carcinoma

DNA amplification is associated with genomic instability, the main characteristic of cancer cells, and it frequently involves protooncogenes. Double minute chromosomes (DM) and homogeneously stained regions (HSR) are cytological manifestations of DNA amplification. Gain of chromosome 19 is a recurrent alteration in mouse hepatocellular carcinoma (HCC). In one tumor cell line established from HCC developed in myc transgenic mice, DM derived from chromosome 19 were identified by spectral karyotyping and confirmed by fluorescence in situ hybridization (FISH). A probe generated by PCR from microdissected DM was localized by FISH on normal and HCC-derived cell lines on DM and chromosome 19 at two sites separated by several medium size G-bands. This organization of DM containing amplified sequences from separate loci of the same chromosome, indicates a complex mechanism of DNA amplification, possibly involving more than one gene. DM or HSR were not previously identified in mouse HCC and adult human HCC. The recognition of these loci could lead to the cloning of new genes or identification of known genes important in development or progression of HCC.     

In situ nick translation distinguishes between C-band positive regions on mouse chromosomes

In situ nick translation of mouse metaphase chromosomes by non-radioactive detection means and DNase I digestion followed by Giemsa staining were used to analyse the DNase I resistance of two different C-band positive regions. These were the centromeric heterochromatin of aero- and metacentric chromosomes and an interstitial C- band on chromosome 1 of wild mice, IS(HSR;1C5D)1Lub. Whereas the centromeric heterochromatin was clearly resistant to DNase I, the interstitial C-band showed very high DNase I sensitivity. Among centromeric C-bands, the heterochromatin in Robertsonian fusion biarmed chromosomes was more resistant to DNase I action than was the centromeric heterochromatin of the acrocentric chromosomes.     

A new variant of chromosome 1 in the house mouse

In wild mouse populations of Siberia, animals with a new variant of chromosome 1 were found. The total length of this chromosome was 1.3 times as great as the normal homologue. The G-banding technique revealed two additional insertions Is(HSR; 1C5)1Icg and Is(HSR; 1E3)2Icg located between bands 1C5 and 1D, and 1E3 and 1E4, resp. The C-banding of both the insertions was positive and lighter than that of the centromeric heterochromatin. The size of each insertion was approximately 15% of new variant of chromosome 1. No meiotic disturbances were found in heterozygous male mice. Chromosome 1 with insertions has been introduced into the laboratory mouse stock.     

Replication pattern of a large homogenously staining chromosome region in antifolate-resistant Chinese hamster cell lines

We have investigated the replication pattern of a large, homogenously staining chromosome region (HSR) in two antifolate-resistant Chinese hamster cell lines. This region is believed to be the location of an amplified genetic sequence which includes at least the gene coding for dihydrofolate reductase and which may be present in as many as 200 copies. It is shown that the HSR in both cell lines is among the first chromosome regions to begin DNA synthesis after reversal of an early G1 block. In cells synchronized in the S period with hydroxyurea, it is also clear that the HSR in both cell lines begins replication at many sites within its length in early S. The replicons comprising the HSR therefore may respond to a common initiation signal in early S. In one cell line (A3), replication of the HSR requires, at most, 3 hours of a 7-hour S period; in a second line (MQ19), replication proceeds for approximately 5 hours. In neither line does replication of the HSR occur concomitantly with synthesis of characteristic late replicating regions. These results were confirmed in exponential cultures using a retroactive labeling technique. The significance of these findings is discussed with reference to the possible origin and arrangement of the amplified sequence in these two cell lines.     

Molecular cytogenetic mapping of Humulus lupulus sex chromosomes

Dioecy is relatively rare in plants and sex determination systems vary among such species. A good example of a plant with heteromorphic sex chromosomes is hop (Humulus lupulus). The genotypes carrying XX or XY chromosomes correspond to female and male plants, respectively. Until now no clear cytogenetic markers for the sex chromosomes of hop have been established. Here, for the first time the sex chromosomes of hop are clearly identified and characterized. The high copy sequence of hop (HSR1) has been cloned and localized on chromosomes by fluorescence in situ hybridization. The HSR1 repeat has shown subtelomeric location on autosomes with the same intensity of the signal. The signal has been present in the subtelomeric region of the long arm and in the near-centromeric region but absent in the telomeric region of the short arm of the X chromosome. At the same time the signal has been found in the telomeric region only of the long arm of the Y chromosome. This finding indicates that the sex chromosomes of hop have evolved from a pair of autosomes via ancient translocation or inversion. The observation of the meiotic configuration of the sex bivalents shows the location of a pseudoautosomal region on the long arms of X and Y chromosomes.     

Gene mapping on maize pachytene chromosomes by in situ hybridization

A sensitive in situ hybridization technique which was effective for mapping genes of low copy number on human metaphase chromosomes was used for gene mapping on maize pachytene chromosomes. A cloned genomic EcoR1 fragment of 10.8 kb, containing most or all of the sequence encoding the Waxy locus mRNA, was used as the probe. Southern DNA blotting analyses performed by Shure et al. (1983) indicated that the Waxy locus was a single copy sequence. In our in situ hybridization experiment, the probe hybridized to a specific site on chromosome 9. Labeling at this site was detected in 48.6% of 154 randomly selected copies of chromosome 9. To test the sensitivity of the method, subclones of the fragment with insert sizes of 6.6, 4.7, 3.5, 2.3, 1.9 and 0.8 kb were used for in situ hybridizations. Labeling efficiency for each probe was determined. The data showed that a single copy probe of 1.9 kb could be detected at the correct position in 18% of 183 randomly selected number 9 chromosomes.     

Double insertion of homogeneously staining regions in chromosome 1 of wild Mus musculus musculus: effects on chromosome pairing and recombination

An examination of the meiotic pattern of chromosome 1 isolated from a feral mouse population and containing a double insertion (Is) of homogeneously staining regions (HSRs) was carried out. The region delineated by the proximal breakpoint of Is(HSR;1C5) 1Icg and the distal breakpoint of Is(HSR;1E3)2Icg is desynapsed during the early pachytene stage and heterosynapsed at the midpachytene, as shown by electron microscopic analysis of synaptonemal complexes. The HSRs have no effect on the segregation of chromosome 1 in heterozygous mice. The lack of homosynapsis in the region under study causes chiasmata redistribution in heteromorphic bivalents. In normal males, single chiasmata are located in the medial part of the chromosome. In heterozygotes, this segment is heterosynapsed and unavailable for recombination. This leads to a significant decrease in the frequency of bivalents bearing single chiasmata. The total number of chiasmata per bivalent is much higher in heterozygous males than in normal ones. The recombination frequency between proximal markers fz and In also is higher in heterozygous animals. The increase in the total chiasma number in the heteromorphic bivalent is due to the addition of double chiasmata located mostly at precentromeric and pretelomeric regions of the chromosome.     

The causes of appearance of a double insertion of homogeneously staining regions in the chromosome 1 of house mouse (Mus musculus musculus)]

A high resolution analysis of G-band pattern of normal and aberrant chromosome 1 bearing two linked insertions of homogeneously staining regions (HSRs) in the house mouse (Mus musculus musculus) reveals an inverted pattern of the euchromatic region between the HSRs. On the basis of this analysis, a hypothesis on the causes for appearance of the aberrant chromosome was put forward: the double insertion is a result of inversion of the chromosome 1 of Mus musculus domesticus bearing a single long insertion. The proximal breakpoint is localized inside the HSR and the distal one--between subbands E3 and E4. From the point of view of these data, new symbols for the aberrations are proposed: Ls (HSR, 1C5) 1Icg--for the proximal insertion, Is(HSR, 1D)21cg--for the distal one, In (1) 1Icg--for the inverted region, including the bands D, E1-E3 and the insertion Is(HSR 1D)21cg.     

Evidence for bacterial origin of heat shock RNA-1

The heat shock RNA-1 (HSR1) is a noncoding RNA (ncRNA) reported to be involved in mammalian heat shock response. HSR1 was shown to significantly stimulate the heat-shock factor 1 (HSF1) trimerization and DNA binding. The hamster HSR1 sequence was reported to consist of 604 nucleotides (nt) plus a poly(A) tail and to have only a 4-nt difference with the human HSR1. In this study, we present highly convincing evidence for bacterial origin of the HSR1. No HSR1 sequence was found by exhaustive sequence similarity searches of the publicly available eukaryotic nucleotide sequence databases at the NCBI, including the expressed sequence tags, genome survey sequences, and high-throughput genomic sequences divisions of GenBank, as well as the Trace Archive database of whole genome shotgun sequences, and genome assemblies. Instead, a putative open reading frame (ORF) of HSR1 revealed strong similarity to the amino-terminal region of bacterial chloride channel proteins. Furthermore, the 5′ flanking region of the putative HSR1 ORF showed similarity to the 5′ upstream regions of the bacterial protein genes. We propose that the HSR1 was derived from a bacterial genome fragment either by horizontal gene transfer or by bacterial infection of the cells. The most probable source organism of the HSR1 is a species belonging to the order Burkholderiales.     

Chromosomal localization of human endogenous retroviral element ERV1 to 18q22----q23 by in situ hybridization

From a clone containing the entire locus of human endogenous retroviral element ERV1, we have obtained a DNA probe that is specific for the 3' long terminal repeat (LTR) sequence. This probe was used to map the LTR of ERV1 by in situ hybridization to chromosomes from normal human blood lymphocytes. The LTR was found to be localized to the distal portion of the long arm of human chromosome 18, within bands q22----q23. This chromosome locus is near the constitutive fragile site at band q21.3 on chromosome 18 associated with the 14;18 translocations seen in follicular lymphomas.     

Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM ± 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.     

Loss and stabilization of aminopterin resistance in murine cell lines

Mouse L-cell lines (B-82, tk-) were obtained using the stepwise selection method, their aminopterin (AP) resistance being 10(3)-5 X 10(4) times higher than that of parental cells. This resistance increase results from dihydrofolate reductase (DHFR) gene amplification which was determined from the 15-120-fold rise of the enzyme activity and with the cytogenetical techniques. The development and loss of AP resistance have been studied and karyological analysis of the lines obtained carried out. Two types of karyological changes were found in stable DM and HSR cells which correspond to extrachromosomal and intrachromosomal forms of the amplified material organization. Localization of the DHFR gene in HSR was proved using the in situ hybridization technique. Extrachromosomal localization of the amplified genes in DM providing unstable AP resistance is dominant at the early stages of the development of resistance and for a long time. It was demonstrated that DM and HSR can exist in one cell during the prolonged period. DHFR gene copy number in such cells is regulated by a change in the DM number, whereas the HSR size and localization are highly stable. HSR covers 1.7-1.9% of the genome length and 38-40% of the marker chromosome length. The genes localized in HSR provide stable AP resistance. Evidence on some intermediate, relative stabilization of the resistance has been obtained. This stabilization is mediated by temporary integration of DHFR copies into other chromosomal sites, in addition to HSR.     

Genetic and physical mapping of barley telomeres

Barley (Hordeum vulgare L.) telomeres were investigated by means of pulsed field gel electrophoresis (PFGE) and in situ hybridization. In situ hybridization showed that a tandemly repeated satellite sequence has a subtelomeric location, and is present at thirteen of the fourteen chromosome ends. PFGE revealed that this satellite sequence is physically close to the telomeric repeat. Pulsed field gel electrophoresis was then used for segregation analysis and linkage mapping of several telomeric and satellite loci in a segregating doubled-haploid population. The telomeric repeat displayed a hypervariable segregation pattern with new alleles occurring in the progeny. Eight satellite and telomeric sites were mapped on an restriction fragment length polymorphism (RFLP)-map of barley, defining the ends of chromosome arms 1L, 2S, 3L, 4S, 4L, 5S and 6. One satellite locus mapped to an interstitial site on the long arm of chromosome 3. The pyhsical location of this locus was confirmed by in situ hybridization to wheat/barley addition line 3.     

Nonradioactive in situ nick translation combined with counterstaining: Characterization of C-band and silver positive regions in mouse testicular cells

The DNase I sensitivity of three different chromatin regions in mouse testicular cells was analysed by in situ nick translation with biotin-dUTP combined with various counterstaining techniques. The regions were: (i) the constitutive centromeric heterochromatin, (ii) an interstitial C-band positive insertion on chromosome 1, Is(HSR1;C5)1Lub, and (iii) the chromatin containing rDNA (designated nucleolar chromatin herein). Incorporated biotin was detected either by the horseradish peroxidase reaction with diaminobenzidine (DAB) or the alkaline phosphatase reaction with fast red. The latter resulted in a water insoluble red precipitate, which was easily removable by any organic solution thus allowing the application of various counterstaining protocols. DNase I sensitivity of the three chromatin regions was screened in different cell types of the mouse testis. The interstitial Is(HSR) region was highly DNase I sensitive when it was recognizable by strong mithramycin fluorescence. The centromeric heterochromatin was DNase I resistant when it was compacted into microscopically visible chromosomal structures (mitosis, pachytene, metaphase I and II). In interphase nuclei from Sertoli cells and spermatogonia it became highly DNase I sensitive. In round spermatids it displayed medium DNase I sensitivity. Nucleolar chromatin was not labelled by in situ nick translation when silver staining demonstrated strong protein production. Sperm cells were highly DNase I sensitive from stages 11 to 15, but resistant as mature spermatozoa.     

Polymorphic HSRs in chromosome 1 of the two semispecies Mus musculus musculus and M. m. domesticus have a common origin in an ancestral population

HSRs (homogeneously staining regions) are the cytological correlates of DNA amplification. In the house mouse, Mus musculus, many populations are polymorphic for the presence or absence of HSRs on chromosome 1. In the semispecies M. m. domesticus the amplified DNA is present within one HSR, whereas in M. m. musculus chromosomes 1 with two HSRs are found. Hybridization of HSR-specific probes to Southern blots of HSR-carrying genomic DNAs from different localities and semispecies revealed similar complex band patterns. the remaining variation is restricted to sequences with a low degree of amplification. Variation is higher between semispecies than within one semispecies. It is assumed that HSRs are derived from one original amplification event and that unequal recombination is the mechanism underlying the length variation of HSRs present today in both semispecies. Evidence from G-banding and in situ hybridization shows that the two HSRs of M. m. musculus originated from a single HSR by means of a paracentric inversion, where one break-point was located within the single HSR and the second outside the HSR. As a consequence of the paracentric inversion the two HSRs of M. m. musculus are permanently linked together. Since exchange of genes between the two semispecies is restricted to a narrow hybrid zone the amplification that gave rise to the HSR most probably occurred prior to the divergence into the semispecies M. m. domesticus and M. m. musculus about 1 million years ago.by D. Schweizer     

Myogenin is in an evolutionarily conserved linkage group on human chromosome 1q31-q41 and unlinked to other mapped muscle regulatory factor genes

Myogenin is a member of a family of muscle-specific regulatory factors which includes MyoD1, Myf-5, and Myf-6 (also called MRF4 and herculin). Extensive regions of sequence homology in genes for these three factors suggest duplication events associated with their evolution. In the present study, the chromosomal location of the myogenin gene in humans (MYOG), mice (Myog), and Chinese hamsters (MYOG) was determined using in situ hybridization to human metaphase chromosomes as well as segregation analysis among interspecific somatic cell hybrid panels and interspecific backcrossed mice. We localize the gene encoding myogenin to human chromosome 1q31-q41 within a linkage group homologous with a region on mouse chromosome 1 and Chinese hamster chromosome 5. The results verify the nonlinkage of MYOG to MYOD1, MYF5, and MYF6 genes and indicate that events associated with the duplication of MYOG with respect to MYOD1, MYF5, or MYF6 loci were not chromosome-wide.     

Cytogenetic analysis of retinoblastoma: evidence for multifocal origin and in vivo gene amplification

Retinoblastoma (Rb) is an uncommon childhood tumor of the neural retina with a significant genetic component in its etiology. A small proportion of patients have a deletion in chromosome 13 encompassing band 13q14, an observation which permitted the assignment of the RB1 locus to this region. About 20% of Rb tumors exhibit microscopic deletions of band 13q14 or monosomy 13. Trisomy 1q and i(6p) have also been reported in a high percentage of tumors. We analyzed the chromosome complements from direct preparations of 10 Rb tumors derived from seven patients. Modal chromosome numbers ranged from 45 to 48, and occasional duplications of the genomes were noted. In general, the tumors were chromosomally stable, although karyotypic evolution and random chromosome loss were encountered. Consistent abnormalities included trisomy 1q, i(6p), 6q-, and del(13)(q12----14). One patient with bilateral Rb had three tumor clones (two in one eye and one in the other) with chromosome abnormalities unrelated in origin. A second patient with unilateral Rb had two tumor clones with chromosome abnormalities again unrelated in origin. These two patients provide some of the first cytogenetic evidence for the multifocal origin of primary Rb. In the untreated tumor of a third patient, a homogeneously staining region (HSR) was detected in 1p32, indicating gene amplication in vivo; previously, an HSR at this site has been reported in the established Rb cell line Y79.