Genomic sequence sampling: a strategy for high resolution sequence-based physical mapping of complex genomes.

We present a simple and efficient method for constructing high resolution physical maps of large regions of genomic DNA based upon sampled sequencing. The physical map is constructed by ordering high density cosmid contigs and determining a sequence fragment from each end of every clone. The resulting map, which contains 30-50% of the complete DNA sequence, allows the identification of many genes and makes possible PCR amplification of virtually any part of the genome. We apply this strategy to the automated analysis of the genome of the primitive eukaryote Giardia lamblia and evaluate its applicability to the physical mapping and DNA sequencing of the human genome.

A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias.

Some acute lymphocytic leukaemias, particularly those in young children, are associated with a t(4;11)(q21;q23) reciprocal translocation. We have cloned the translocation breakpoint on chromosome 11q23 and isolated corresponding RNA transcripts from this region. The translocation occurs within a cluster of Alu repetitive elements located within an intron of a gene that gives rise to 11.5 (kb) transcript spanning the translocation breakpoint. The 11.5 kb transcript encodes a protein that is highly homologous to the Drosophila trithorax gene, a developmental regulator. An analysis of a series of leukaemic patients carrying t(4;11) and t(9;11) translocations indicate that the majority of breakpoints in infant leukaemias lie within a 5 kb region.

The EXT2 multiple exostoses gene defines a family of putative tumour suppressor genes.

Hereditary multiple exostoses (EXT) is an autosomal dominant condition characterized by short stature and the development of bony protuberances at the ends of all the long bones. Three genetic locl have been identified by genetic linkage analysis at chromosomes 8q24.1, 11p11-13 and 19p. The EXT1 gene on chromosome 8 was recently identified and characterized. Here, we report the isolation and characterization of the EXT2 gene. This gene shows striking sequence similarity to the EXT1 gene, and we have identified a four base deletion segregating with the phenotype. Both EXT1 and EXT2 show significant homology with one additional expressed sequence tag, defining a new multigene family of proteins with potential tumour suppressor activity.

Alterations of the PPP2R1B gene in human lung and colon cancer.

The PPP2R1B gene, which encodes the beta isoform of the A subunit of the serine/threonine protein phosphatase 2A (PP2A), was identified as a putative human tumor suppressor gene. Sequencing of the PPP2R1B gene, located on human chromosome 11q22-24, revealed somatic alterations in 15% (5 out of 33) of primary lung tumors, 6% (4 out of 70) of lung tumor-derived cell lines, and 15% (2 out of 13) of primary colon tumors. One deletion mutation generated a truncated PP2A-Abeta protein that was unable to bind to the catalytic subunit of the PP2A holoenzyme. The PP2R1B gene product may suppress tumor development through its role in cell cycle regulation and cellular growth control.

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.

Integrating maps of human chromosome 11.

The past year has seen major advances in our understanding of chromosome structure, driven by technology that allows the rapid construction of physical and genetic maps. Information on the structure and organization of human chromosome 11 is rapidly being accumulated as a result of these developments.

Physical mapping of the human genome by pulsed field gel analysis.

Detailed physical maps of large regions of the human genome are important for locating and cloning genes responsible for human hereditary diseases, as well as for obtaining a more detailed understanding of chromosome structure and evolution. Pulsed field gel electrophoresis provides one method for generating physical maps of non-methylated rare restriction endonuclease sites. This review summarizes recent progress in the isolation of region-specific mapping probes and in their application for the physical mapping of selected regions of the human genome.

Interphase cytogenetics for the detection of the t(11;22)(q24;q12) in small round cell tumors.

Among the small round cell tumors differential diagnosis is particularly difficult for their undifferentiated or primitive character. In this mixed group of tumors, only the primitive neuroectodermal tumors, which include Ewing's sarcoma (ES), show the unique and consistent feature of the (11;22)(q24;q12) translocation, which can therefore be considered a hallmark of these neoplasias. We analyzed four primitive neuroectodermal tumor cell lines, one osteosarcoma cell line, and 11 patients by fluorescent in situ hybridization with cosmid clones 23.2 and 5.8, bracketing the t(11;22) at 11q24. Metaphase spreads from tumor cell lines, and from biopsy specimens of three patients with ES were analyzed. In the remaining eight patients comprising five ES, two small cell osteosarcomas and one chronic osteomyelitis, only nuclei preparations were available for analysis. We detected the t(11;22) in interphase nuclei of the four primitive neuroectodermal tumor cell lines, of three patients in which the karyotype demonstrated the translocation and in five cases of ES in which cytogenetic analysis had not been possible. Two cases of small cell osteosarcoma and one chronic osteomyelitis were also analyzed and were both normal with respect to the t(11;22). By analyzing cell lines and small round cell tumor samples by fluorescent in situ hybridization, we established that interphase cytogenetics is a rapid alternative to chromosomal analysis for the detection of the t(11;22) and represents an invaluable tool for the differential diagnosis of small round cell tumors.

EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations.

We have determined the frequency of EWS fusion transcripts in a series of primary Ewing's sarcomas and peripheral primitive neuroectodermal tumors and cells lines. Type 1 and 2 EWS-Fli1 fusions were demonstrated in 8 cell lines and 14 patient samples. Five patients with cytogenetically characterized rearrangements of chromosome 22 that did not involve chromosome 11 were included in these studies. A novel EWS-Fli1 in-frame isoform fusing EWS to exon 8 of Fli1 was isolated from a tumor with a variant t(12;22;22)(q14;p1;q12) translocation. Three in-frame isoforms of a novel hybrid transcript derived from the fusion of EWS with the ETS domain of the human erg gene were identified in patient samples and a cell line with cytogenetically unidentified or cryptic translocations involving chromosomes 21 and 22. Interphase analysis by fluorescent in situ suppression hybridization using two overlapping erg yeast artificial chromosome clones demonstrated disruption of the erg gene on chromosome 21 in a patient sample with monosomy 22. Our results provide new information about the involvement of EWS in small round cell tumors involving exchange of its putative RNA-binding domain with DNA-binding domains derived from different members of the ETS family of transcription factors. These studies emphasize the utility of reverse transcriptase PCR analysis and fluorescent in situ hybridization as additional diagnostic tools for differential diagnosis among small round cell tumors.

Characterization of two atypical promoters and alternate mRNA processing in the mouse Thy-1.2 glycoprotein gene.

The promoter and 5' flanking region of the mouse Thy-1.2 glycoprotein gene were characterized by DNA sequencing, primer extension analysis, and deletion analysis. Transcriptional initiation sites were identified which corresponded to two separate exons upstream of the portion of the gene encoding the Thy-1.2 glycoprotein. We demonstrated that the mouse Thy-1.2 gene was transcribed from two atypical promoters separated by 260 base pairs in the genomic sequence. These promoters contained neither TATAAG nor GGPyCCAATCT homologous sequences but defined a conserved nonamer CTCCCTGCT at -48 from each initiation site. Two Thy-1.2 mRNA species of 1,835 and 1,939 nucleotides, differing in the 5' untranslated region of the mRNA, were thus transcribed from the single Thy-1.2 gene by mRNA splicing to the same downstream exon. Recombinant genomes in which the bacterial chloramphenicol acetyltransferase gene was expressed from either of the two Thy-1.2 promoters demonstrated that each promoter functioned independently and did not direct cell-specific expression in lymphoid cells. The 5' flanking region of the Thy-1.2 gene upstream of -68 could be eliminated without altering cell-type-specific expression. This suggests that regulatory elements responsible for tissue and developmental stage-specific expression of the Thy-1.2 gene are not present in the 5' flanking DNA but may reside downstream of the promoters.

Novel molecular approach to define pest species status and tritrophic interactions from historical Bemisia specimens.

Museum specimens represent valuable genomic resources for understanding host-endosymbiont/parasitoid evolutionary relationships, resolving species complexes and nomenclatural problems. However, museum collections suffer DNA degradation, making them challenging for molecular-based studies. Here, the mitogenomes of a single 1912 Sri Lankan Bemisia emiliae cotype puparium, and of a 1942 Japanese Bemisia puparium are characterised using a Next-Generation Sequencing approach. Whiteflies are small sap-sucking insects including B. tabaci pest species complex. Bemisia emiliae's draft mitogenome showed a high degree of homology with published B. tabaci mitogenomes, and exhibited 98-100% partial mitochondrial DNA Cytochrome Oxidase I (mtCOI) gene identity with the B. tabaci species known as Asia II-7. The partial mtCOI gene of the Japanese specimen shared 99% sequence identity with the Bemisia 'JpL' genetic group. Metagenomic analysis identified bacterial sequences in both Bemisia specimens, while hymenopteran sequences were also identified in the Japanese Bemisia puparium, including complete mtCOI and rRNA genes, and various partial mtDNA genes. At 88-90% mtCOI sequence identity to Aphelinidae wasps, we concluded that the 1942 Bemisia nymph was parasitized by an Eretmocerus parasitoid wasp. Our approach enables the characterisation of genomes and associated metagenomic communities of museum specimens using 1.5 ng gDNA, and to infer historical tritrophic relationships in Bemisia whiteflies.

Combined femoral and Chiari osteotomies for reconstruction of the painful subluxation or dislocation of the hip in cerebral palsy. A long-term outcome study.

In order to treat painful subluxation or dislocation secondary to cerebral palsy, 11 patients (12 hips) underwent combined femoral and Chiari pelvic osteotomies with additional soft-tissue releases at a mean age of 14.1 years (9.1 to 17.8). Relief of pain, improvement in movement of the hip, and in sitting posture, and ease of perineal care were recorded in all, and were maintained at a mean follow-up of 13.1 years (8 to 17.5). The improvement in general mobility was marginal, but those who were able to walk benefited the most. The radiological measurements made before operation were modified afterwards to use the lateral margin of the neoacetabulum produced by the pelvic osteotomy. The radiological migration index improved from a mean of 80.6% (61% to 100%) to 13.7% (0% to 33%) (p < 0.0001). The mean changes in centre edge angle and Sharp's angle were 72 degrees (56 degrees to 87 degrees; p < 0.0001) and 12.3 degrees (9 degrees to 15.6 degrees; p < 0.0001), respectively. Radiological evidence of progressive arthritic change was seen in one hip, in which only a partial reduction had been achieved, and there was early narrowing of the joint space in another. Painless heterotopic ossification was observed in one patient with athetoid quadriplegia. In seven hips the lateral Kawamura approach, elevating the greater trochanter, provided exposure for both osteotomies and allowed the construction of a dome-shaped iliac osteotomy, while protecting the sciatic nerve.

Identification of a 6-cM minimal deletion at 11q23.1-23.2 and exclusion of PPP2R1B gene as a deletion target in cervical cancer.

Previous functional and deletion mapping studies on cervical cancer (CC) have implicated one or more tumor suppressor genes (TSGs) on chromosome 11 at q13 and q22-24 regions. Of these, the 11q22-24 region exhibits frequent allelic deletions in a variety of solid tumor types, suggesting the presence of critical genes for tumor suppression in this region. However, the precise region of deletion on 11q is not clearly defined in CC. In an attempt to accurately map the deleted region, we performed an extensive loss of heterozygosity (LOH) mapping in 58 tumors using 25 polymorphic loci on both the short and long arms. The pattern of LOH identified three sites of deletions, two on 11p (p15.11-p15.3 and p12-13), and one on 11q (q23.1-q23.2). The 11q23.1-q23.2 exhibited highest frequency (60.6%) of deletions, suggesting that this could be the site of a candidate TSG in CC. The minimal deletion at 11q23.1-23.2 was restricted to a 6-cM region between 123.5 and 129.5 cM genetic distance on chromosome 11, identifying the site of a potential TSG important in the pathogenesis of CC. At least five known genes and 28 UniGene clusters were mapped to the present commonly deleted region. In addition, we have excluded a previously known TSG PPP2R1B at 11q23 as a deletion target in CC. The definition of the minimal deletion and the availability of expressed sequence resources should facilitate the identification of the candidate TSG.

Loss of heterozygosity in sporadic human breast carcinoma: a common region between 11q22 and 11q23.3.

The development of sporadic human breast cancer is associated with the accumulation of genetic alterations on several chromosomes. In the case of chromosome 11, loss of heterozygosity (LOH) at loci on the short arm has been well documented and suggests the presence of a suppressor gene(s) at 11p15.5. However, the evidence for similar events on the long arm is less compelling. Here, we determined the prevalence of LOH for chromosome 11q in 44 malignant and 3 benign cases of unselected sporadic breast tumor samples. We found that alteration of chromosome 11q is common in the pathogenesis of breast cancer as 19 of 44 (43%) malignant tumor specimens exhibited LOH. Eleven (58%) of these genetic alterations were specific to the long arm of the chromosome. The smallest region of shared LOH places the target between 11q22 and 11q23.3, the same general region frequently altered in cancers of the ovary, colon, skin, and uterine cervix, perhaps indicating the location of a tumor suppressor gene or genes of importance in each of these different tumor types.

Loss of heterozygosity for chromosome 11 in primary human breast tumors is associated with poor survival after metastasis.

A common feature of the malignant progression of human tumors is loss of heterozygosity (LOH) for various regions of their genomes. Such events encompassing chromosomes 11p15 and 11q23 are frequent in human breast tumors. Here, we have analyzed genetic and clinical characteristics of a series of primary breast tumors in order to determine: (a) a more finely mapped estimate of the involved regions; (b) whether there is a relationship in the presentation of LOH between the two regions; and (c) whether a correlation exists between such LOH and any of the clinical parameters pertaining to each patient. We found that LOH for 11p15.5 and 11q23 occurred in 35 and 46% of the 86 primary breast carcinomas, respectively, but in none of the 10 benign tumors examined. The minimal region of LOH for 11p15 was in the approximately 2-megabase region between loci TH and D11S988. Twenty-nine % of the tumors showed LOH simultaneously at both 11p15 and 11q23, 5% had LOH only at 11p15.5, and 15% had LOH only at 11q23. Among these genetic groups, clinical features such as tumor size, involvement of auxiliary nodes, histological subtype, tumor grade, estrogen/progesterone receptor status, and patient age were not markedly different. However, LOH of 11q23 (either alone or in conjunction with LOH of 11p15) in the primary tumor was found to be highly predictive of aggressive postmetastatic disease course with substantially reduced survival (P = 0.0004; log rank test). We also observed a slight trend toward a more rapid development of metastatic lesions, without obvious site specificity, in patients with primary tumors showing LOH for chromosome 11 in the pathogenesis of human breast cancer; we suggest that its effects are late in the progression of this disease.

Inhibitor of protein synthesis co-isolating with polyribosomal RNA.

Although low concentrations of total polyribosomal RNA from porcine parotid glands or rat pituitary cells in culture (GH3) isolated by standard dodecylsulphate/phenol, chloroform extraction techniques can direct the incorporation of radiolabeled amino acids into proteins using a cell-free protein-synthesizing system derived from wheat germ embryos, higher concentrations inhibit the translation of endogenous wheat germ mRNA, or added rabbit globin mRNA or polyribouridylic acid. This inhibitory activity is separated from poly(A)-rich RNA by oligo(dT)-cellulose chromatography. The inhibitory activity appears to reside in a heat-stable protein since it is inactivated by incubation with various proteases but not by DNAase I, pancreatic ribonuclease, alkaline hydrolysis, or treatment with formamide. Specificity of the inhibition is suggested since the inhibitory fraction prepared from GH3 cells also inhibits protein synthesis in a cell-free protein-synthesizing system derived from porcine parotid gland, while the inhibitory fraction prepared from porcine parotid gland has no inhibitory activity in this homologous system. Radioiodination and dodecylsulphate/polyacrylamide gel electrophoresis reveal several protein bands, the most prominent with an apparent molecular weight of 78 000.

Human trithorax gene rearrangements in therapy-related acute leukaemia after etoposide treatment.

Molecular analysis of leukaemic blasts from 9 patients with secondary myeloid leukaemia reveals rearrangements of the human trithorax gene (Htrx-1) in three patients, including one in whom abnormalities of chromosome 11 band q23 were not detected by conventional cytogenetics. All three patients had been treated with epipodophyllotoxins, whilst none of the six without rearrangements had received these agents. The patients with rearrangements also presented with different clinical features. These findings support the separation of secondary leukaemia into two classes, and correlate rearrangements of the Htrx-1 gene with a group of secondary leukaemias that follow specific cancer treatment regimens.

Preprolactin represents the initial product of prolactin mRNA translation.

mRNA isolated from a line of functional rat pituitary tumor cells (GH3) added to a cell-free protein synthesizing system derived from wheat embryo directed the biosynthesis of only one protein which is immunoprecipitated by prolactin antiserum. This protein, 2000--3,000 daltons heavier than prolactin and referred to as preprolactin, appeared to represent the initial, authentic product of translation since [35S]methionine donated by initiator Met-tRNAi was documented to be incorporated into the N-terminal portion; the molar ratio of leucine: initiator methionine was estimated to be 25--28:1, while prolactin contains 22 leucine residues. The mRNA coding for preprolactin was demonstrated to migrate at approximately 12--13S using rate zonal centrifugation. These data are compatible with the postulate that preprolactin represents the intact, initial product of prolactin mRNA translation.