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Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Peroxisome proliferator-activated receptor binding protein (PBP), a nuclear receptor coactivator, interacts with estrogen receptor alpha (ERalpha) in the absence of estrogen. This interaction was enhanced in the presence of estrogen but was reduced in the presence of antiestrogen, tamoxifen. Transfection of PBP in CV-1 cells resulted in enhancement of estrogen-dependent transcription, indicating that PBP serves as a coactivator in ER signaling. To examine whether overexpression of PBP plays a role in breast cancer because of its coactivator function in ER signaling, we determined the levels of PBP expression in breast tumors. High levels of PBP expression were detected in approximately 50% of primary breast cancers and breast cancer cell lines by ribonuclease protection analysis, in situ hybridization, and immunoperoxidase staining. Fluorescence in situ hybridization of human chromosomes revealed that the PBP gene is located on chromosome 17q12, a region that is amplified in some breast cancers. We found PBP gene amplification in approximately 24% (6/25) of breast tumors and approximately 30% (2/6) of breast cancer cell lines, implying that PBP gene overexpression can occur independent of gene amplification. This gene comprises 17 exons that, together, span >37 kilobases. The 5'-flanking region of 2.5 kilobase pairs inserted into a luciferase reporter vector revealed that the promoter activity in CV-1 cells increased by deletion of nucleotides from -2,500 to -273. The -273 to +1 region, which exhibited high promoter activity, contains a typical CCAT box and multiple cis-elements such as C/EBPbeta, YY1, c-Ets-1, AP1, AP2, and NFkappaB binding sites. These observations, in particular PBP gene amplification, suggest that PBP, by its ability to function as ERalpha coactivator, might play a role in mammary epithelial differentiation and in breast carcinogenesis.

Loss of a whole chromosome 5 or a deletion of the long arm, del(5q), is a recurring abnormality in malignant myeloid diseases. In previous studies, we delineated a commonly deleted segment of approximately 4 Mb within band 5q31 that was flanked by IL9 on the proximal side and D5S166 on the distal side. We have generated a physical map of P1 (PAC), bacterial (BAC), and yeast artificial chromosome (YAC) clones of this interval. The contig consists of 108 clones (78 PACs, 2 BACs, and 28 YACs) to which 125 markers (5 genes, 11 expressed sequence tags, 12 polymorphisms, and 97 sequence-tagged sites) have been mapped. Using PAC clones for fluorescence in situ hybridization analysis of leukemia cells with a del(5q), we have narrowed the commonly deleted segment to 1-1.5 Mb between D5S479 and D5S500. To search for allele loss, we used 7 microsatellite markers within and flanking the commonly deleted segment to examine leukemia cells from 28 patients with loss of 5q, and 14 patients without cytogenetically detectable loss of 5q. In the first group of patients, we detected hemizygous deletions, consistent with the cytogenetically visible loss; no homozygous deletions were detected. No allele loss was detected in patients without abnormalities of chromosome 5, suggesting that allele loss on 5q is the result of visible chromosomal abnormalities. The development of a stable PAC contig and the identification of the smallest commonly deleted segment will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 5q.

We have generated a physical map of human chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The yeast artificial chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Peroxisomal acyl-CoA oxidase (ACOX; EC 1.3.3.6) is the first enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, and it donates electrons directly to molecular oxygen, thereby producing H2O2. The discovery of carcinogenic peroxisome proliferators, which markedly increase the levels of this H2O2-producing ACOX in rat and mouse liver, generated interest in peroxisomal beta-oxidation system genes. The present study deals with the structural organization of human ACOX gene. This gene spans approximately 33 kb and consists of 14 exons and 13 introns. Primer-extension analysis revealed three principal cap sites, which were mapped at 50, 52, and 53 nt upstream of the initiator methionine codon. The 5' flanking region of the ACOX gene was sequenced up to 500 bp upstream of the cap sites. This promoter region is G + C-rich and contains three copies of the "GC box" hexanucleotides. Multiple GC boxes are a characteristic feature of the rat ACOX and bifunctional protein genes of the beta-oxidation system. A + T-rich TATA-boxlike sequences, TTTATTT and TTATT, have also been identified in this human ACOX gene, but typical CCAAT motifs are absent. This ACOX gene has been mapped to chromosome 17q25 by in situ hybridization, using a biotinlabeled probe.

Loss of a whole chromosome 5 or a deletion of its long arm (5q) is a recurring abnormality in malignant myeloid neoplasms. To determine the location of genes on 5q that may be involved in leukemogenesis, we examined the deleted chromosome 5 homologs in a series of 135 patients with malignant myeloid diseases. By comparing the breakpoints, we identified a small segment of 5q, consisting of band 5q31, that was deleted in each patient. This segment has been termed the critical region. Distal 5q contains a number of genes encoding growth factors, hormone receptors, and proteins involved in signal transduction or transcriptional regulation. These include several genes that are good candidates for a tumor-suppressor gene, as well as the genes encoding five hematopoietic growth factors (CSF2, IL3, IL4, IL5, and IL9). By using fluorescence in situ hybridization, we have refined the localization of these genes to 5q31.1 and have determined the order of these genes and of other markers within 5q31. By hybridizing probes to metaphase cells with overlapping deletions involving 5q31, we have narrowed the critical region to a small segment of 5q31 containing the EGR1 gene. The five hematopoietic growth factor genes and seven other genes are excluded from this region. The EGR1 gene was not deleted in nine other patients with acute myeloid leukemia who did not have abnormalities of chromosome 5. By physical mapping, the minimum size of the critical region was estimated to be 2.8 megabases. This cytogenetic map of 5q31, together with the molecular characterization of the critical region, will facilitate the identification of a putative tumor-suppressor gene in this band.

Chromosomal translocations involving chromosome 3, band q27, are among the most common rearrangements in B-cell non-Hodgkin lymphoma. From a bacteriophage lambda library prepared from a lymphoma characterized by a t(3;14)(q27;q32), genomic clones were isolated using a probe from the immunoglobulin heavy chain locus (IGH) joining region. In addition to clones containing an apparently normal IGH rearrangement, others were found to contain one of the translocation breakpoint junctions. Normal chromosome 3 sequences and the reciprocal breakpoint junction were subsequently isolated. DNA probes on each side of the chromosome 3 breakpoint hybridized at high stringency to the DNA of various mammalian species, demonstrating evolutionary conservation. One such probe from the presumptive der(3) chromosome detected an 11-kilobase transcript when hybridized to RNA of B- and T-cell lines. A probe made from partial cDNA clones isolated from a T-cell line hybridized with genomic DNA from both sides of the chromosome 3 breakpoint, indicating that the t(3;14) is associated with a break within the gene on chromosome 3. In situ chromosomal hybridization revealed that the same gene is involved in the t(3;22)(q27;q11). Preliminary nucleotide sequencing shows no identity of the cDNA to gene sequences in available data banks. We propose the name BCL6 (B-cell lymphoma 6) for this gene, since it is likely to play a role in the pathogenesis of certain B-cell lymphomas.

An important family of regulatory molecules is made up of proteins that possess the DNA-binding and dimerization motif known as the basic helix-loop-helix (bHLH) domain. The bHLH family includes subgroups of closely related proteins that share common functional properties and overlapping patterns of expression (e.g., the MyoD1 and achaete-scute subgroups). In this report we describe HEN1 and HEN2, mammalian genes that encode a distinct subgroup of bHLH proteins. The HEN1 gene was identified on the basis of cross-hybridization with TAL1, a known bHLH gene implicated in T-cell acute lymphoblastic leukemia. In situ fluorescence hybridization was used to localize the human HEN1 gene to chromosome band 1q22. HEN1 and HEN2 are coexpressed in the IMR-32 human neuroblastoma cell line, and they encode highly related proteins of 133 and 135 residues, respectively, that share 98% amino acid identity in their hHLH domains. These data imply that the bHLH protein subgroup encoded by HEN1 and HEN2 may serve important regulatory functions in the developing nervous system.

Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells.

In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3) are the three members of the neurotrophin family known to exist in mammals. Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4), which shares all of the features found in the mammalian neurotrophins, has been identified in Xenopus and viper. We used sequences specific to the Xenopus/viper NT-4 to isolate a neurotrophin from both human and rat genomic DNA that appears to represent the mammalian counterpart of Xenopus/viper NT-4. Human NT-4 as well as a human NT-4 pseudogene colocalize to chromosome 19 band q13.3. Mammalian NT-4 has many unusual features compared to the previously identified neurotrophins and is less conserved evolutionarily than the other neurotrophins. However, mammalian NT-4 displays bioactivity and trk receptor specificity similar to that of Xenopus NT-4.

Tumor-specific alteration of the TAL1 gene occurs in almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL). We now report the identification of TAL2, a distinct gene that was isolated on the basis of its sequence homology with TAL1. The TAL2 gene is located 33 kilobase pairs from the chromosome 9 breakpoint of t(7;9)(q34;q32), a recurring translocation specifically associated with T-ALL. As a consequence of t(7;9)(q34;q32), TAL2 is juxtaposed with sequences from the T-cell receptor beta-chain gene on chromosome 7. TAL2 sequences are actively transcribed in SUP-T3, a T-ALL cell line that harbors the t(7;9)(q34;q32). The TAL2 gene product includes a helix-loop-helix protein dimerization and DNA binding domain that is especially homologous to those encoded by the TAL1 and LYL1 protooncogenes. Hence, TAL2, TAL1, and LYL1 constitute a discrete subgroup of helix-loop-helix proteins, each of which can potentially contribute to the development of T-ALL.

Recurring chromosomal translocations involving chromosome 11, band q23, have been observed in acute lymphoid leukemias and especially in acute myeloid leukemias. We recently showed that breakpoints in four 11q23 translocations, t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13.3), were contained within a yeast artificial chromosome clone bearing the CD3D and CD3G gene loci. We have identified within the CD3 yeast artificial chromosome a transcription unit that spans the breakpoint junctions of the 4;11, 9;11, and 11;19 translocations, and we describe two other, related transcripts that are upregulated in the RS4;11 cell line. We have named this gene MLL (myeloid/lymphoid, or mixed-lineage, leukemia.

Fragile sites are specific regions of chromosomes that are prone to breakage. In cells cultured under conditions that induce fragile site expression, high levels of inter- and intrachromosomal recombination have been observed involving chromosomal bands containing fragile sites. To determine whether expression of specific fragile sites would facilitate preferential integration of exogenous DNA at these recombination hot spots, the vector pSV2Neo was transfected into a Chinese hamster-human somatic cell hybrid containing a derivative chromosome 3 as its only human component. Chromosome 3 contains a common fragile site at band 3p14.2 (FRA3B) that is induced by aphidicolin. Both cells induced to express FRA3B and the uninduced control cells were transfected with the pSV2Neo selectable plasmid. In situ hybridization of a biotin-labeled pSV2Neo probe to metaphase chromosomes revealed one to three integration sites in each stably transfected clone. Four of 13 clones transfected under conditions of FRA3B induction showed integration of pSV2Neo at 3p14; these clones also showed specific integration into hamster chromosome 1 and a rearranged chromosome characteristic of CHO cells (mar2). The 7 control clones, however, showed an apparently random pattern of pSV2Neo integration. Significant hybridization of pSV2Neo to both FRA3B and Chinese hamster chromosomes 1 and mar2 was seen in 100 cells from pooled colonies transfected after treatment with aphidicolin. These results suggest that preferential integration of marker DNA into human and Chinese hamster fragile sites occurs with exposure to aphidicolin. The nature of the DNA sequences at fragile sites is unknown and, despite a number of approaches, these sequences have not yet been isolated; our procedure may represent an approach to the cloning of fragile sites.

Translocations involving chromosome 11, band q23, are frequent recurring abnormalities in human acute lymphoblastic and acute myeloid leukemia. We used 19 biotin-labeled probes derived from genes and anonymous cosmids for hybridization to metaphase chromosomes from leukemia cells that contained four translocations involving band 11q23: t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13). The location of the cosmid probes relative to the breakpoint in 11q23 was the same in all translocations. Of the cosmid clones containing known genes, CD3D was proximal and PBGD, THY1, SRPR, and ETS1 were distal to the breakpoint on 11q23. Hybridization of genomic DNA from a yeast clone containing yeast artificial chromosomes (YACs), that carry 320 kilobases (kb) of human DNA including CD3D and CD3G genes, showed that the YACs were split in all four translocations. These results indicate that the breakpoint at 11q23 in each of these translocations occurs within the 320 kb encompassed by these YACs; whether the breakpoint within the YACs is precisely the same in the different translocations is presently unknown.