Dynamic increase of a 45,X cell line in a patient with multicentric ring Y chromosomes

Because ring Y chromosomes are unstable during cell division most reported patients are mosaics, usually including a 45,X cell line. The phenotype varies from normal males or females with streak gonads to sexual ambiguities. We present here the case of a 23-year-old man who was referred at 11 years for growth delay. The GTG-banded karyotypes of lymphocytes revealed two cell lines: 46,X,dic r(Y) seen in 76% of the metaphases analyzed and 45,X (24%). Karyotypes and FISH were performed eight years later with the following probes: DYZ3 (Y centromere), SRY (sex-region of the Y), DYZ1 (Yq heterochromatin), CEPX/Y (X centromere and Yq heterochromatin), TelVysion Xp/Yp, Xq/Yq (X and Y subtelomeres), pan-telomeric, cosmid clones LLycos130G04 and LLycos37C09 (PARII), and BAC clone RP11-5C5 (Yq11.223). The results showed an increase in the 45,X cell line (60%) and a reduction in the 46,X,dic r(Y) cell line (36.4%). The use of Yq probes showed that the ring Y chromosome was dicentric. In addition, other ring Y structures were observed. The breakpoints occurred in proximal Yp11.32 or in Yp11.31 distal to SRY and in Yq12 distal to the PARII region. Therefore, most of the Y remained intact and all genes, with the exception of those in PARI, are present in double dosage in the dic r(Y). The level of mosaicism was important in defining the phenotype.     

47,X,idic(Y),inv dup(Y): a non-mosaic case of a phenotypically normal boy with two different Y isochromosomes and neocentromere formation

A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)(n). The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.     

A possible association of Y chromosome heterochromatin with stature

Summary A correlation between Y chromosome length and stature was statistically analyzed in a normal male population of 142 Japanese students with a mean age of 24.0 years. Evidence was obtained that increased length of the heterochromatic band Yq12 may be associated with increased height: The correlation coefficient between band Yq12 length and height was 0.17, statistically significant at the 5% level. And, taller males had longer Y chromosomes, in which the mean length of band Yq12 was significantly longer than that of shorter males. No correlation was seen between length of the euchromatic band Yq11 and stature. The present study reveals a possible effect of Yq heterochromatin on the development of body height in man.     

Selective protection of specific DNA sequences in the heterochromatin of C-banded human Y chromosomes.

The molecular basis of C-banding was investigated by in situ hybridization of human Y chromosome-derived repeated sequences, DYZ1 and DYZ2, to untreated or to alkaline-treated metaphases. Autoradiography of G-banded metaphases showed that both probes hybridized to the long arm of Y. Alkaline hydrolysis significantly reduced grain number for DYZ2 (58%-82%; P less than .05) but not for DYZ1 (P greater than .05). Similar results were observed for interphase nuclei. These findings demonstrated that the heterochromatin of the long arm contains at least two repetitive DNA fractions having two different sensitivities to alkaline hydrolysis. These observations support the notion that DYZ2 maps terminally on the Yq arm and may be nonheterochromatic.     

Distribution of DYZ2 repetitive sequences on the human Y chromosome

The distribution of the 2000 copies of the Y-specific repetitive family DYZ2 is controversial since previous reports have mapped these sequences to different sites of the Yqh region. In this work, we have performed non-radioactive in situ hybridization of a cloned DYZ2 fragment at higher stringency conditions on 5-aza-cytidine-enlarged Y chromosomes; the results suggest a non-uniform distribution of these sequences, which are preferentially located at the proximal and distal parts of Yqh, including the C+/Q-heterochromatin at the boundary with the euchromatic region.     

Nonfluorescent Y chromosomes. Cytologic evidence of origin

Summary Twelve presumptive structurally altered Y chromosomes were studied with Q-, G-, G-11, C-, Cd, and lateral asymmetric banding techniques and were compared with normal X and Y chromosmes and with an abnormal [i(Yq)] Y chromosome that exhibited intact fluorescence. Significant to this work is the fact that the Y chromosome has a small block of Giemsa-11 heterochromatin adjacent to the centromere on the long arm, while the X chromosome does not, which allows a distinction between the X-and Y-derived chromosomes. Two of the twelve altered chromosomes of either X or Y origin are small nonfluorescent rings. Each ring has a G-11-positive band of heterochromatin at the centromere, confirming Y origin. Each of the normal-length nonfluorescent presumed Ys and a Y with a fluorescent band in the center have one G-11 band at the centromere and another at an equal distance from the end of the long arm, the bands also being Cd positive, indicating that these chromosomes are pseudodicentric. The likely mechanism of origin is a break at the distal bright heterochromatin/ euchromatin junction (or within the bright segment in the chromosome with the bright center band), fusion of the sister chromatids at the breakpoints, and loss of the distal segment.     

Molecular detection of a translocation (Y;11)(q11.2;q24) in a 45,X male with signs of Jacobsen syndrome

Summary A 45,X karyotype was found in a boy with dysmorphic features, hypoglycaemia and pancytopenia. DNA analysis showed the presence of the Y-chromosomal DNA sequences SRY, ZFY, DYZ4, DYZ3 and DYS1. Using fluorescent in situ hybridization, we located DYZ4 and DYZ3 on chromosome llqter and concluded that a de novo translocation (Y;11)(q11.2;q24) with a deletion of 11q24qter and a deletion of Yq11.2Yqter were present; Jacobsen syndrome and azoospermia are associated with these deletions. Signs of Jacobsen syndrome were observed in the patient.     

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes

Summary In situ hybridization experiments were carried out with two clones, YACG 35 and 2.8, which had been selected from two genomic libraries strongly enriched for the human Y chromosome. Besides the human Y chromosome, both sequences strongly hybridized to the human X chromosome, with few minor binding sites on autosomes. In particular, on the X chromosome DNA from clone YACG 35 hybridized to the centromeric region and the distal part of the short arm (Xp2.2). On the Y chromosome, the sequence was assigned to one site situated in the border region between Yq1.1 and Yq1.2. DNA from clone 2.8 also hybridized to the centromeric region of the X and the distal part of the short arm (Xq2.2). On the Y, however, two binding sites were observed (Yp1.1 and Yq1.2). The findings indicate that sex chromosomal sequences may be localized in homologous regions (as suggested from meiotic pairing) but also at ectopic sites.     

Interstitial deletions of repetitive DNA blocks in dicentric human Y chromosomes

Cytogenetic analysis of aberrant human Y chromosomes was done by fluorescence in situ hydbridization (FISH) with Y specific repetitive DNA probes. It revealed an interstitial deletion of different DNA blocks in two dicentric chromosome structures. One deletion includes the total alphoid DNA structure of one centromeric region. The second deletion includes the total repetitive DYZ5 DNA structure in the pericentromeric region of one short Y arm. Both dicentric Y chromosomes were iso(Yp) chromosomes with break and fusion point located in Yq11, the euchromatic part of the long Y arm. Their phenotypic appearance was abnormal, resembling small monocentric Yq-chromosomes in metaphase plates. Mosaic cell lines, usually included in karyotypes with dicentric Y chromosomes, were not observed. It is assumed that both deletion events suppress the kinetochore activity in one Y centromeric region and thus stabilize its dicentric structure. Local interstitial deletion events had not been described in dicentric human Y chromosomes, but are common in dicentric yeast chromosomes. This raises the question of whether deletion events in dicentric human chromosomes are rare or restricted to the Y chromosome or also represent a general possibility for stabilization of a dicentric chromosome structure in human.     

Molecular analysis of aberrations of Xp and Yq

Summary Three cases of Y chromosomal aberrations were studied using a panel of Y-specific DNA sequences from both Yp and euchromatic Yq. One case was a phenotypic male fetus with a Y-derived marker chromosome. The short arm of this chromosome was intact, but most of its long arm was missing. The second case had a 46,Xyq- karyotype with portions of euchromatic Yq, including the spermatogenesis region, missing. The third case was a phenotypic female with a 46,XXp+ karyotype. The extra material on the Xp+ chromosome was derived from the heterochromatic, and part of the euchromatic, portion of Yq. Application of X-specific DNA sequences demonstrated that the distal portion of the short arm of the translocation X chromosome was deleted (Xpter—p22.3). The three examples demonstrate the importance of diagnostic DNA analysis in cases of marker chromosomes, and X and Y chromosomal aberrations. In addition, the findings in the patients facilitate further deletion mapping of euchromatic Yq.     

Organizational variation of DYZ1 repeat sequences on the human Y chromosome and its diagnostic potentials

The long arm of the human Y chromosome is flecked with various fractions of repetitive DNA. DYZ1 is one such fraction, which is organized tandemly as an array of a 3.4-kb repeat ranging from 2000-4000 copies in normal males. We have studied the organizational variation of the DYZ1 fraction on the human Y chromosome using DNA samples from CEPH family members and the random population employing the RFLP approach, fluorescence in situ hybridization (FISH), and conducted a similarity search with GenBank sequences. Typing of genomic DNA using DYZ1 as a probe showed an allele length and copy number variations even between two male siblings. Hybridization of DNA from monochromosome hybrids with this probe showed its presence on chromosome 15 in addition to the Y chromosome. Fluorescence in situ hybridization of metaphase chromosomes from an apparently normal male showed DYZ1 sequences in the proximal region of chromosome 11 in addition to the long arm of the Y chromosome. Typing of sets of semen and blood DNA samples from the same human individuals showed discernible allelic variation between the two samples, indicating tissue-specific programmed sequence modulation. DYZ1 seems to be the first probe having the unique potential to discriminate unequivocally the difference between the DNA originating from semen and blood samples, and may be exploited in forensic cases. This probe may also be used as a diagnostic tool to ascertain Y chromosome mosaicism in patients (e.g., Turner), its aberrant status in somatic cells, and possible sequence modulation/rearrangement in the germline samples. Additionally, this can be used to uncover sequence polymorphism in the human population.     

Types and frequencies of Q-variant chromosomes in a Japanese population

Summary The types of Q-variant bands were determined by a combination of numerical designations setting five levels for both the size of bands and the intensity of fluorescence. This scoring system was used in a study of the frequencies of Q variants in 400 Japanese individuals: variant bands were observed in seven specific autosome pairs of Nos. 3,4,13,14,15,21, and 22. The number of variants per individual ranged from 0 to 8, and the mean was 3.83±1.86. The incidence of Q variants according to the types of variant bands was determined in specific chromosomes.A low frequency of No. 3 chromosome variants and a high frequency of a long Y in males seems to be characteristic for Japanese populations.Variation in the length of the long arm of Y (Yq) was analyzed in a total of 157 men. The relative length of Yq, which was determined by a ratio of Yq/21q, ranged from 0.98 to 2.27, with an average of 1.56±0.25. The length of pale band Yq11 was relatively constant between individuals, with an average of 0.64±0.08. Therefore, it was clear that the variation in the Yq length was the result mainly of a variation in the length of the brilliant band Yq12. However, a slight tendency for the length of band Yq11 to increase in proportionally to the total length of the Yq was revealed. In this study special consideration was paid to the reliable analysis of Q-band heteromorphism, and the factors or obstacles preventing such analysis have been discussed briefly.     

DA/DAPI-Fluorescent heteromorphism of human Y chromosome

Summary Variation of DA/DAPI intensity in the Yq12 band was observed in five amniotic cell specimens and one blood specimen from the father of one fetus. Three distinct classes of Yq heterochromatin were identified by distamycin A (DA) treatment of the cell cultures and various staining techniques. The heterochromatin in the Yq11.23 sub-band does not under-condense when exposed to DA, and shows pale fluorescence with quinacrine staining, positive C-banding, and bright fluorescence with DA/DAPI technique. This class of heterochromatin was consistently observed in all specimens studied. The other two classes of heterochromatin are in the Yq12 band. Both show undercondensation when exposed to DA, quinacrine-bright fluorescence, and positive C-banding; howover, one class of heterochromatin shows DA/DAPI-bright fluorescence and the other shows pale fluorescence. The size and banding intensity of the two classes of heterochromatin in Yq12 are variable. These results provide cytological evidence of heterogeneity within the Y heterochromatin region containing AT-rich DNA.     

A method for the rapid sequence-independent amplification of microdissected chromosomal material

We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3' end and a defined sequence at its 5' end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.     

Physical mapping of unique nucleotide sequences on identified rice chromosomes

A physical mapping method for unique nucleotide sequences on specific chromosomal regions was developed combining objective chromosome identification and highly sensitive fluorescence in situ hybridisation (FISH). Four unique nucleotide sequences cloned from rice genomic DNAs, varying in size from 1.3 to 400 kb, were mapped on a rice chromosome map. A yeast artificial chromosome (YAC) clone with a 399 kb insert of rice genomic DNA was localised at the distal end of the long arm of rice chromosome (1q2.1) and a bacterial artificial chromosome (BAC) clone (180 kb) containing the rice leaf blast-resistant gene (Pi-b) was shown to occur at the distal end of the long arm of chromosome 2 (2q2.1). A cosmid (35 kb) with the resistance gene (Xa-21) against bacterial leaf blight was mapped on the interstitial region of the long arm on chromosome 11 (11q1.3). Furthermore a single RFLP marker, 1.29 kb in size, was mapped successfully to the distal region of the long arm of rice chromosome 4 (4q2.1). For precise localisation of the nucleotide sequences within the chromosome region, image analyses were effective. The BAC clone was localised to the specific region, 2q2.1:96.16, by image analysis. The result was compared with the known location of the BAC clone on the genetic map and the consistency was confirmed. The effectiveness and reliability in physically mapping nucleotide sequences on small plant chromosomes achieved by the FISH method using a variety of probes was unequivocally demonstrated.     

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome ΔYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from ΔYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.     

Rate of DNA replication in the DNA synthetic period of the barley chromosomes

The rate of DNA replication, as judged by H³-thymidine incorporation, at the specific time of the S-period in chromosomes of barley (Hakata No. 2) is studied by means of autoradiography.In the barley chromosomes, two different DNA units with respect to replication-time are distinguishable. The early replicating DNA is replicated at least within 1 hour ab init. of the S-period, and the late replicating DNA within 1/2 to 1 hour before the end of the S-period. The replication scarcely occurs in the middle of the S-period. These evidences suggest that the replication of chromosomal DNA in the present material does, therefore, not proceed in a continuous time sequence. Topographically, the early replicating DNA is almost confined exclusively to the distal regions of the chromosomes 1 and 5, and this situation seems applicable to other chromosomes as well, whereas the late replicating DNA is close to the centromere on its both sides. Hence, the replication of chromosomal DNA does not proceed uniformly in a longitudinal sequence along the chromosomes. The interrelationships among chromosome structure in its cytological expression, replication -pattern and -time of chromosomes, and regulating mechanisms of DNA replication are discussed.     

Huntington disease-linked restriction fragment length polymorphism localized within band p16.1 of chromosome 4 by in situ hybridization.

A 5.5-kilobase (kb) single sequence DNA fragment (G8) reveals the DNA polymorphic locus D4S10 on Southern blot analysis. This locus is closely linked to Huntington disease and has been mapped to chromosome 4 short arm using human-mouse somatic cell hybrids, and specifically to chromosome 4 band p16 using DNA from individuals with deletions of chromosome 4 short arm who exhibit Wolf-Hirschhorn syndrome. With in situ hybridization techniques, we have confirmed the location of D4S10 on chromosome 4 and further localized it within band p16 utilizing five patients, four with overlapping chromosome 4 short-arm aberrations. The DNA segment G8 was hybridized to the mataphase chromosomes of the five patients. Two of them have different interstitial deletions of one of the chromosome 4 short arms (TA and BA), two have different chromosome 4 short-arm terminal deletions (RG and DQ), and one has a normal male karyotype. By noting the presence or absence of hybridization to the partially deleted chromosomes with known precise breakpoints, we were able to more accurately localize probe G8 to the distal half of band p16.1 of chromosome 4.     

Mouse paracentric inversion In(3)55Rk mutates the urate oxidase gene

The paracentric inversion In(3)55Rk on mouse Chromosome 3 (Chr 3) was induced by cesium irradiation. Genetic crosses indicate the proximal breakpoint cosegregates with D3Mit324 and D3Mit92; the distal breakpoint cosegregates with D3Mit127, D3Mit160, and D3Mit200. Giemsa-banded chromosomes show the inversion spans approximately 80% of Chr 3. The proximal breakpoint occurs within band 3A2, not 3B as reported previously; the distal breakpoint occurs within band 3H3. Mice homozygous for the inversion exhibit nephropathy indicative of uricase deficiency. Southern blot analyses of urate oxidase, Uox, show two RFLPs of genomic mutant DNA: an EcoRI site between exons 4-8 and a BamHI site 3' to exon 6. Mutant cDNA fails to amplify downstream of base 844 at the 3' end of exon 7. FISH analysis of chromosomes from inversion heterozygotes, using a cosmid clone containing genomic wild-type DNA for Uox exons 2-4, shows that a 5' segment of the mutated Uox allele on the inverted chromosome has been transposed from the distal breakpoint region to the proximal breakpoint region. Clinical, histopathological, and Northern analyses indicate that our radiation-induced mutation, uox(In), is a putative null.     

Identification of a telomeric DNA sequence in Plasmodium berghei.

A fragment of Plasmodium berghei DNA was cloned using a technique designed to select for telomeric sequences. The cloned fragment recognizes Bal31-sensitive bands in P. berghei genomic digests. It contains at its distal end at least 70 tandem repeats of the heptanucleotide sequence CCCTGAAA. The presence of natural single strand discontinuities in the telomeric regions of P. berghei DNA is demonstrated by the selective incorporation of deoxyribonucleoside triphosphates in the absence of DNase. The number of copies of the cloned sequence present in each genome agrees with an estimate of 6-12 chromosomes per nucleus.