Su(UR)ES: A gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)sc^V2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34.8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form “weak points,” underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally β-heterochromatic, acquire a distinct banding pattern, i.e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional α-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.     

The ets sequence from the transforming gene of avian erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21: both loci are transcriptionally active.

Human DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be closely related to the viral equivalent by hybridization and partial sequence analysis. The transforming gene of E26 has a tripartite origin with the structure delta gag [1.2 kilobases (kb) from the viral gag gene]-myb(0.9 kb from the chicken myb gene)-ets (1.6 kb from the chicken ets gene). Human ets DNA is located on two distinct human chromosomes. The human ets-1 locus on chromosome 11 encodes a single mRNA of 6.8 kb; the second locus, ets-2 on chromosome 21, encodes three mRNAs of 4.7, 3.2, and 2.7 kb. The ets-related sequences of human DNA on chromosomes 11 and 21 are discontiguous, except for a small overlap region encoding 14 amino acids, where 12 are conserved between these two loci. By contrast, the chicken homolog has contiguous ets-1 and ets-2 sequences and is primarily expressed in normal chicken cells as a single 7.5-kb mRNA. We conclude that the ets sequence shared by the virus, the chicken, and humans is likely to contain at least two dissociable functional domains, ets-1 and ets-2. Thus, the tripartite transforming gene of E26 includes four distinct domains that may be functionally relevant for the transforming function of the virus (delta gag, myb, ets-1, and ets-2).     

Analysis of a T-cell tumor-specific breakpoint cluster at human chromosome 14q32.

Cytogenetic abnormalities involving chromosome 14 band q32 are consistently observed in human T-cell tumors. Patients with ataxia-telangiectasia (AT) are especially prone to development of these tumors, which frequently carry either inversion inv(14)(q11;q32) or translocation t(14;14) (q11;q32) chromosomes. We have previously shown that the cytogenetic breakpoints of one t(14;14)(q11;q32) chromosome and two inv(14)(q11;q32) chromosomes in T-cell tumors from AT and non-AT patients join the T-cell receptor alpha chain locus, at chromosome band 14q11, with a region(s) at 14q32 centromeric of the immunoglobulin heavy chain variable region (VH) gene IGHV. We now show that these two inv(14) breakpoints are linked by 2.1 kb of germ-line 14q32 DNA and that the three breakpoints define, by in situ hybridization analysis, a single locus at chromosome band 14q32.1 located about 15-20 million base pairs on the centromeric side of the IGH locus. Sequence analysis of the 14q32.1 breakpoint regions indicates that abnormal recombination does not universally result from mistaken V-D-J joining (D = diversity region; J = joining region). Therefore, we invoke a tumor selection model to describe the role of the 14q32.1 locus in tumor development.     

Location of the 18/28S ribosomal RNA genes in two Hawaiian Drosophila species by monoclonal immunological identification of RNA.DNA hybrids in situ.

Using both heterologous rabbit antisera and mouse monoclonal antibody to RNA.DNA hybrids, we have mapped the in situ hybridization locus of the 18/28S ribosomal RNA fraction to a single large band on polytene autosome 3 in Drosophila heteroneura and Drosophila silvestris. This portion of the chromosome is not physically connected with the nucleolus at the end of larval salivary gland development. In mature larvae, little or no hybridization with the material in the nucleolus can be detected. In younger larvae, hybridization of the ribosomal RNA probe to the nucleolus itself can be observed. The chromosome 3 locus is the only band in the polytene genome that shows variation in size and intensity of staining between populations and species. The interband chromosome regions that are immediately distal or proximal to the 18/28S rRNA locus have been involved in a disproportionately large number of natural inversion breaks observed in the euchromatic portion of the polytene chromosome. In 104 species of Hawaiian Drosophila in which chromosome 3 polytene sequences have been determined, 15 breaks occur in these two regions. On a random basis, only one such break is expected. We propose that this locus may be flanked by substantial heterochromatic blocks which are not represented in the salivary gland chromosome.     

Isolation and Chromosomal Localization of Highly Repeated DNA Sequences in Drosophila melanogaster

The nuclear DNA of D. melanogaster contains DNA sequences that are repeated between ten and a hundred times more often than the next class of redundant DNA. This DNA, as a renatured duplex isolated on the basis of its renaturation kinetics, has a buoyant density of 1.691 (g/ml). In its native state it bands within the unique nuclear DNA peak (rho = 1.701). These sequences have been localized by "in situ" hybridization in the chromocenter of the chromosomes of the salivary gland. The properties of centromeres are discussed in terms of the occurrence of repeated sequences at this locus.     

High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.     

Cloning and characterization of a dispersed, multicopy, X chromosome sequence in Drosophila melanogaster.

We have isolated and characterized a dispersed middle repetitive DNA sequence from Drosophila melanogaster that is concentrated on the euchromatic portion of the X chromosome. In situ hybridization of the repeat unit to salivary gland chromosomes shows the sequence is distributed among approximately 10 major and 20 minor X chromosomal sites. Based on DNA sequence analysis of homologous sequences from three different cytogenetic regions, the 372-base-pair repeat unit appears to be (A + T)-rich and noncoding and shows strong sequence conservation among units from different chromosomal regions. The nature and distribution of this sequence are suggestive of the hypothetical X chromosome DNA sequences thought to be involved in the primary establishment of sex determination and dosage compensation in Drosophila.     

Molecular limits on the size of a genetic locus in Drosophila melanogaster.

This report places outer limits on the size of the DNA region required for expression of a Drosophila gene. This region, termed the unit of expression, includes not only the structural gene but also any cis-acting sequences that modulate its activity. The locus we have chosen, Sgs-4, codes for one of the glue proteins secreted by larval Drosophila salivary glands. Cytological deficiencies have been identified that eliminate sequences on one side or the other of Sgs-4 without affecting its expression. The ends of these deficiencies have been localized accurately with respect to restriction endonuclease sites in and near the locus. These endpoints limit the Sgs-4 structural gene and essential flanking sequences to a 16- to 19-kilobase region of the X chromosome. The results also show that there is no DNA sequence rearrangement in the Sgs-4 region during development of either the polytene larval salivary glands or adult flies.     

Refinement of lymphoma cytogenetics by the chromosome 18q21 major breakpoint region

A small (2.8-kilobase, kb) major breakpoint region localized to segment 18q21 rearranges in greater than 70% of t(14;18)(q32;q21) lymphomas. This rearrangement interrupts the Bcl-2 gene and introduces it into the Ig locus at 14q32. The rearrangement between the joining region (JH) of Ig on chromosome 14 and the 18q21 region creates a translocation- specific DNA rearrangement. We generated probes that distinguish the 14;18 juncture on the derivative (der) 14 and der (18) chromosomes, providing a molecular approach to t(14;18) identification. Approximately 60% of unselected follicular lymphomas, 20% of diffuse large cell lymphomas, and 50% of adult undifferentiated non-Burkitt lymphomas demonstrated 14;18 rearrangements within the major breakpoint region. Examination of DNA for 14;18 rearrangements resolved the identity of 14q+ chromosomes in two patient's cells that lacked an obvious reciprocal partner. Identification of the exact restriction fragments that mediate translocations complements routine cytogenetics. The detection of DNA rearrangements does not require dividing cells or the presence of an identifiable reciprocal partner and can detect clonal translocation rearrangements when the neoplastic cells are only a minority of all cells present.     

Spontaneous mutation rates at enzyme loci in Drosophila melanogaster.

In a marked-inversion-balanced lethal system mutations were accumulated at a minimum pressure of natural selection on 2000 second chromosomes of Drosophila melanogaster that originated from 4 stem chromosomes. Five enzyme loci were tested: alpha-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), malate dehydrogenase (Mdh, EC 1.1.1.37), alcohol dehydrogenase (EC 1.1.1.1), hexokinase-C (Hex-C tentative name), and alpha-amylase (Amy, EC 3.2.1.1). Three band-morph mutants, one at the Mdh locus, one at the Hex-C locus, and one at the Amy locus, were detected out of 1,658,308 allele replications. In addition, 17 null mutants were found. Accepting that the number of structural genes is the same as that of bands in the salivary gland chromosomes, the total mutation rate per generation for all the structural genes in the second chromosomes is estimated to be 0.008-0.040, which is much smaller than that estimated for viability polygenes (0.12-0.17). Thus, it is speculated that most viability and other fitness polygenes are located in controlling regions outside the structural genes.     

Glycoprotein tumour markers in head and neck neoplasms —a consecutive study on CA-50, CA 19-9, and CEA

Summary Serum levels of three glycoprotein tumour antigens (carcino-embryonic antigen, CEA; cancer-associated antigen 50, CA-50; gastrointestinal cancer-associated antigen, CA 19-9) were determined on 125 consecutive patients with tumours of the head and neck region. Elevated CEA values (> 5 units/ml) were found in 13/70 squamous cell carcinomas, 3/21 benign and 4/18 malignant salivary gland neoplasms. Elevated CA-50 values (> 17 units/ml) were found in 19/70 squamous cell carcinomas, 6/18 malignant and 1/21 benign salivary neoplasms. CA 19-9 displayed higher values (> 37 units/ml) in 9/68 squamous cell carcinomas, 4/18 malignant and none of 21 benign salivary gland tumours. Combination of CEA and CA-50 analyses increased the proportion of elevated values to 30/70 in squamous cell carcinomas and 10/18 in salivary gland malignancies. In squamous cell carcinomas no correlation between staging or grading and serum levels was detected for any of the markers. Among malignant salivary gland tumours, CA-50 displayed enhanced serum values in 4/6 mucoepidermoid carcinomas. The mean values for CA-50 and CA 19-9 serum levels were significantly higher for malignant salivary gland neoplasms compared to benign tumours. There was a close correlation between CA-50 and CA 19-9 serum levels. Although, the results suggest that at present none of the tumour markers tested have a place alone in the routine examination of patients with tumours affecting the head and neck region, further studies on salivary gland neoplasms and combinations of the tumour markers are justified.This study was supported by grants from the Swedish Society for Cancer Research and Lions Research Foundation, Umeå Sweden     

Gene amplification in Rhynchosciara salivary gland chromosomes.

Late in the fourth larval instar, several regions of the Rhynchosciara americana salivary gland chromosomes undergo "DNA puffing." We have constructed a library of cloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands during the period of development when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puff sites C3 and C8 and encode 1.25-kilobase and 1.95-kilobase RNA molecules, respectively. We estimate from the quantitation of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing.     

Transformation of salivary gland secretion protein gene Sgs-4 in Drosophila: stage- and tissue-specific regulation, dosage compensation, and position effect.

The Sgs-4 gene of Drosophila melanogaster encodes one of the larval secretion proteins and is active only in salivary glands at the end of larval development. This gene lies in the X chromosome and is controlled by dosage compensation--i.e., the gene is hyperexpressed in males. Therefore, males with one X chromosome produce nearly as much Sgs-4 products as females with two X chromosomes. We used a 4.9-kilobase-pair (kb) DNA fragment containing the Sgs-4d coding region embedded in 2.6 kb of upstream sequences and 1.3 kb of downstream sequences for P-element-mediated transformation of the Sgs-4h underproducer strain Kochi-R. Sgs-4d gene expression was found in all 15 transformed lines analyzed, varying with the site of chromosomal integration. The transposed gene was subject to tissue- and stage-specific regulation. At X-chromosomal sites, the levels of gene expression were similar in both sexes, signifying dosage compensation. At autosomal sites, it was on average 1.5 times higher in males than in females. The results indicate that the transforming DNA fragment contains all sequences necessary for tissue- and stage-specific regulation and for hyperexpression in males.     

流式细胞学DNA分析在良、恶性涎腺多形性腺瘤中的应用价值

目的　探讨流式细胞学 DNA分析 (FCM)对良、恶性涎腺多形性腺瘤的临床价值。方法　采用流式细胞仪测定 5 0例良性涎腺多形性腺瘤、18例恶性涎腺多形性腺瘤和 10例正常涎腺石蜡包埋组织的 DNA倍体及细胞增殖活性 ,分析其变化与预后的关系。结果　正常涎腺组织 DNA皆为二倍体 ;良性涎腺多形性腺瘤 DNA二倍体率为 84 % (42例 ) ,异倍体率 16 % (8例 ) ;恶性涎腺多形性腺瘤分别为 6 1% (11例 )、39% (7例 ) ,与良性者比较有显著性差异 (P<0 .0 5 )。结论　 FCM诊断良、恶性涎腺多形性腺瘤具有一定价值 ,其应用应与形态学检查相结合。