The “Spot 14” gene resides on the telomeric end of the 11q13 amplicon and is expressed in lipogenic breast cancers: Implications for control of tumor metabolism

Enhanced long chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. “Spot 14” (S14) is a carbohydrate- and thyroid hormone-inducible nuclear protein specific to liver, adipose, and lactating mammary tissues that functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 11q13, where the S14 gene (THRSP) resides, also predicts a poor prognosis in breast tumors. We localized the S14 gene between markers D11S906 and D11S937, at the telomeric end of the amplified region at 11q13, and found that it was amplified and expressed in breast cancer-derived cell lines. Moreover, concordant expression of S14 and a key lipogenic enzyme (acetyl-CoA carboxylase) in a panel of primary breast cancer specimens strongly supported a role for S14 as a determinant of tumor lipid metabolism. S14 expression provides a pathophysiological link between two prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification.

A widely expressed βIII spectrin associated with Golgi and cytoplasmic vesicles

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the β-spectrin gene family by homology searches of the GenBank databases and by 5′ rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called βIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. βIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human βIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human βIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that βIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and β-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with βIII spectrin. βIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Fluorescence in situ hybridization analysis of keratinocyte growth factor gene amplification and dispersion in evolution of great apes and humans

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15–21.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q21, 9p11, 9q12–13, 18p11, 18q11, 21q11, and 21q21.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and 21. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.

Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.

Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets

5′-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) of wheat (Triticum aestivum) were cloned and sequenced. The sequences of the two genes, Acc-1,1 and Acc-1,2, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by Acc-1,1 and Acc-1,2 is 93% identical with the maize plastid ACCase but only 80–84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5′-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the Acc-1 genes is at least 130–170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes Acc-1,1 and Acc-1,2, indicates that all three genes are transcriptionally active.

Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders.

Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD). Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma. other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis. To date, the critical genomic segment and candidate genes involved in these deletions have not been identified. In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization. As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3. In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1). Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment. Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster. This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD.

Development of a bovine X chromosome linkage group and painting probes to assess cattle, sheep, and goat X chromosome segment homologies.

The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We have developed bovine chromosome painting probes for Xp and Xq to assess segment homologies between the submetacentric bovine X chromosome and the acrocentric sheep and goat X chromosomes. These painting probes and their corresponding DNA libraries were developed by chromosome micromanipulation, DNA micropurification, microcloning, and PCR amplification. The bovine Xp painting probe identified an interstitially located homologous segment in the sheep and goat Xq region, most probably resulting from chromosome inversion. Ten type II (microsatellite) markers obtained from the bovine Xq library and five other X chromosome assigned, but unlinked, markers were used to generate a linkage map for Xq spanning 89.4 centimorgans. The chromosome painting probes and molecular markers generated in this study would be useful for comparative mapping and tracing of internal X chromosome rearrangements in all ruminant species and would contribute to the understanding of mammalian sex chromosome evolution.

Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix.

We have chosen tumors of the uterine cervix as a model system to identify chromosomal aberrations that occur during carcinogenesis. A phenotype/genotype correlation was established in defined regions of archived, formalin-fixed, and hematoxylin/eosin-stained tissue sections that were dissected from normal cervical epithelium (n = 3), from mild (n = 4), moderate (n = 6), and severe dysplasias/carcinomas in situ (CIS) (n = 13), and from invasive carcinomas (n = 10) and investigated by comparative genomic hybridization. The same tissues were analyzed for DNA ploidy, proliferative activity, and the presence of human papillomavirus (HPV) sequences. The results show that an increase in proliferative activity and tetraploidization had occurred already in mildly dysplastic lesions. No recurrent chromosomal aberrations were observed in DNA extracted from normal epithelium or from mild and moderate dysplasias, indicating that the tetraploidization precedes the loss or gain of specific chromosomes. A gain of chromosome 3q became visible in one of the severe dysplasias/CIS. Notably, chromosome 3q was overrepresented in 90% of the carcinomas and was also found to have undergone a high-level copy-number increase (amplification). We therefore conclude that the gain of chromosome 3q that occurs in HPV16-infected, aneuploid cells represents a pivotal genetic aberration at the transition from severe dysplasia/CIS to invasive cervical carcinoma.

Distribution of haplotypes from a chromosome 21 region distinguishes multiple prehistoric human migrations

Despite mounting genetic evidence implicating a recent origin of modern humans, the elucidation of early migratory gene-flow episodes remains incomplete. Geographic distribution of haplotypes may show traces of ancestral migrations. However, such evolutionary signatures can be erased easily by recombination and mutational perturbations. A 565-bp chromosome 21 region near the MX1 gene, which contains nine sites frequently polymorphic in human populations, has been found. It is unaffected by recombination and recurrent mutation and thus reflects only migratory history, genetic drift, and possibly selection. Geographic distribution of contemporary haplotypes implies distinctive prehistoric human migrations: one to Oceania, one to Asia and subsequently to America, and a third one predominantly to Europe. The findings with chromosome 21 are confirmed by independent evidence from a Y chromosome phylogeny. Loci of this type will help to decipher the evolutionary history of modern humans.

A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2

Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as Gnas, which encodes a G protein α subunit, and there is clinical and biochemical evidence that the human homologue GNAS1, mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes, Gnasxl and Nesp. Gnasxl determines a variant G protein α subunit associated with the trans-Golgi network and Nesp encodes a secreted protein of neuroendocrine tissues. Gnasxl is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas Nesp is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably, Gnasxl, Nesp, and Gnas are all part of the same transcription unit; transcripts for Gnasxl and Nesp are alternatively spliced onto exon 2 of Gnas. This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons.

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

Cladistic association analysis of Y chromosome effects on alcohol dependence and related personality traits

Association between Y chromosome haplotype variation and alcohol dependence and related personality traits was investigated in a large sample of psychiatrically diagnosed Finnish males. Haplotypes were constructed for 359 individuals using alleles at eight loci (seven microsatellite loci and a nucleotide substitution in the DYZ3 alphoid satellite locus). A cladogram linking the 102 observed haplotype configurations was constructed by using parsimony with a single-step mutation model. Then, a series of contingency tables nested according to the cladogram hierarchy were used to test for association between Y haplotype and alcohol dependence. Finally, using only alcohol-dependent subjects, we tested for association between Y haplotype and personality variables postulated to define subtypes of alcoholism—antisocial personality disorder, novelty seeking, harm avoidance, and reward dependence. Significant association with alcohol dependence was observed at three Y haplotype clades, with significance levels of P = 0.002, P = 0.020, and P = 0.010. Within alcohol-dependent subjects, no relationship was revealed between Y haplotype and antisocial personality disorder, novelty seeking, harm avoidance, or reward dependence. These results demonstrate, by using a fully objective association design, that differences among Y chromosomes contribute to variation in vulnerability to alcohol dependence. However, they do not demonstrate an association between Y haplotype and the personality variables thought to underlie the subtypes of alcoholism.

Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation

In Bacillus subtilis, parE and parC were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.