NARWHAL, a primary analysis pipeline for NGS data.

UNLABELLED: The NARWHAL software pipeline has been developed to automate the primary analysis of Illumina sequencing data. This pipeline combines a new and flexible de-multiplexing tool with open-source aligners and automated quality assessment. The entire pipeline can be run using only one simple sample-sheet for diverse sequencing applications. NARWHAL creates a sample-oriented data structure and outperforms existing tools in speed. AVAILABILITY: https://trac.nbic.nl/narwhal/.

Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients.

The cerebral cortex of Alzheimer's and Down syndrome patients is characterized by the presence of protein deposits in neurofibrillary tangles, neuritic plaques, and neuropil threads. These structures were shown to contain forms of beta amyloid precursor protein and ubiquitin-B that are aberrant (+1 proteins) in the carboxyl terminus. The +1 proteins were not found in young control patients, whereas the presence of ubiquitin-B+1 in elderly control patients may indicate early stages of neurodegeneration. The two species of +1 proteins displayed cellular colocalization, suggesting a common origin, operating at the transcriptional level or by posttranscriptional editing of RNA. This type of transcript mutation is likely an important factor in the widely occurring nonfamilial early- and late-onset forms of Alzheimer's disease.

Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli.

We report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host. High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations. No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.COLI: RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.

Isolation of beta-globin-related genes from a human cosmid library.

A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA.

Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells.

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.

A new function of ROD1 in nonsense-mediated mRNA decay.

RNA-binding proteins play a crucial role in the post-transcriptional regulation of gene expression. Polypyrimidine tract binding protein (PTB in humans) has been extensively characterized as an important splicing factor, and has additional functions in 3' end processing and translation. ROD1 is a PTB paralog containing four RRM (RNA recognition motif) domains. Here, we discover a function of ROD1 in nonsense-mediated mRNA decay (NMD). We show that ROD1 and the core NMD factor UPF1 interact and co-regulate an extensive number of target genes. Using a reporter system, we demonstrate that ROD1, similarly to UPF1 and UPF2, is required for the destabilization of a known NMD substrate. Finally, we show through RIP-seq that ROD1 and UPF1 associate with a significant number of common transcripts.

The nucleotide sequence of two restriction fragments located in the gene AB region of bacteriophage S13.

The nucleotide sequence of a double stranded DNA fragment from the gene AB region of bacteriophage S13 DNA has been determined. The fragment was isolated as two adjacent shorter fragments by cleavage of S13 replicative form (RF) DNA with restriction endonuclease III from Hemophilus aegyptius. The strands of the fragments were separated electrophoretically and hydrolyzed with T4 endonuclease IV to yield short oligonucleotides which were then sequenced by partial exonuclease digestion. The complete nucleotide sequence of the restriction fragments was obtained by ordering the inter- and intrastrand overlapping oligonucleotide sequences. The adjacent fragments were 190 nucleotides in length. The sequences included a HindII site, an AluI site and two sequences which may be possible transcription initiation sequences, one with an adjacent sequence homologous to the canonical promoter site sequence T-A-T-Pu-A-T-Pu. Examination of the three possible reading frames for translation of the sequence revealed only one possible complete translation product. The postulated partial sequence of gene A protein has a highly positively charged arginine-rich area which may have importance in DNA binding.

Isolation of transforming DNA by cosmid rescue.

A procedure has been developed to allow the recovery of an integrated plasmid genome from a transformed cell, together with large areas of the flanking DNA sequences. DNA from Saccharomyces cerevisiae BAS2, in which the pBR322--ura 3 plasmid (Y1p5) is integrated at the yeast histone H2A and H2B locus, was used to generate a cosmid library, using a new cosmid vector (pTL5) that is ampicillin sensitive and tetracycline resistant. Colonies were selected for ampicillin resistance, which was conferred by the incorporation of the integrated pBR322 beta-lactamase gene into the recombinant cosmid. Restriction enzyme and blot hybridization analyses show that the rescued clones contain the yeast histone genes in addition to the Y1p5 sequences; a total of approximately 50 kilobase pairs of DNA sequences flanking the plasmid was recovered as a series of overlapping cosmids. This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell.

Fluorescence in situ hybridization analysis of transcript dynamics in cells.

Since the first description of in situ hybridization in 1969 the technique has advanced to allow sensitive detection of DNA and mRNA molecules at the cellular and subcellular levels. In particular fluorescence in situ hybridization (FISH) has become a frequently used tool in basic and applied biomedical research since detection is sensitive and allows discrimination of multiple targets in the same sample. By using RNA-FISH we have been able to detect primary transcripts of the human embryonic, fetal, and adult globins in erythroid cells to study the competitive transcription mechanism or variegated expression patterns of the human beta-globin locus. We have correlated such expression patterns with other parameters such as cell type, cell cycle, replication, and stage of differentiation by simultaneous detection of, e.g., incorporated BrdUTPs, proteins (e.g., cyclins A and E, PCNA, histones), and globin (primary) transcripts and/or locus integration sites. Thus a combination of FISH and immunofluorescence methods allow the visualization of different processes taking place in the nucleus relative to each other in terms of three-dimensional space and structure and time (development, cell cycle).

Alpha-amanitin insensitive transcription of the human epsilon-globin gene.

In vitro transcription was used to show that RNA polymerase III is responsible for the initiation of transcription at a position 200 bp upstream from the epsilon-globin major cap site. High levels of -200 transcription interferes with the RNA polymerase II major cap site transcription. Using DNA mediated transient expression, the ratio of -200 to +1 transcription can be modulated by either the direction of replication or the presence of an enhancing element in the vector. We suggest that this heterogeneous usage of cap sites is not related to epsilon-globin gene transcription in vivo, but is instead the result of a combination of factors inherent to transient expression experiments.

Inactivation of the H-2Klk gene could involve the substitutions of methylated CpGs.

By the isolation of overlapping cosmid clones and 'chromosome walking' studies from the H-2Kk gene, we have obtained cosmid clones encoding the H-2Klk gene from two separate cosmid libraries. The nucleotide sequence of one of the clones was determined. The cloned H-2Klk gene could be transcribed in vitro to give a normal H-2 class I mRNA of 1.7 kb. However, the deletion of four nucleotides in exon 3 of the H-2Klk gene results in a translation termination codon at the beginning of exon 4. In agreement with this, when expressed in human cells, the H-2Klk gene gave a truncated, cytoplasmic polypeptide of Mr 36,000. Therefore, although the H-2Klk gene is homologous to other class I MHC genes in its molecular organization and nucleotide sequence, it is a pseudogene. When compared to the nucleotide sequence of the H-2Kk gene, the H-2Klk gene has undergone many substitutions of methylated CpG residues (meCpG). This represents further evidence to suggest that this gene is inactive.

The sequence GGCmCGG is resistant to MspI cleavage.

MspI essentially fails to cut the sequence GGCmCGG at enzyme concentrations which give total digestion of CCGG, CmCGG and GGCCGG sites. This result explains why certain sites in mammalian DNA are resistant to both MspI and HpaII and shows that this results from an idiosynchracy of MspI rather than a novel form of DNA methylation at this site in mammalian cells.

GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes.

The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain.

Cloning and sequencing of the CRABP-I locus from chicken and pufferfish: analysis of the promoter regions in transgenic mice.

Retinoic acid (RA), a derivative of vitamin A, is an important molecule for development and homeostasis of vertebrate organisms. The intracellular retinoic acid binding protein CRABP-I has a high affinity for RA, and is thought to be involved in the mechanism of RA signalling. CRABP-I is well conserved in evolution and shows a specific expression pattern during development, but mice made deficient for the protein by gene targeting appear normal. However, the high degree of homology with CRABP-I from other species indicates that the protein has been subject to strong selective conservation, indicative of an important biological function. In this paper we have compared the conservation in the expression pattern of the mouse, chicken and pufferfish CRABP-I genes to substantiate this argument further. First we cloned and sequenced genes and promoter regions of the CRABP-I genes from chicken and the Japanese pufferfish, Fugu rubripes. Sequence comparison with the mouse gene did not show any large blocks of homology in the promoter regions. Nevertheless, the promoter of the chicken gene directed expression to a subset of the tissues that show expression with the promoter from the mouse gene. The pattern observed with the pufferfish promoter is even more restricted, essentially to rhombomere 4 only, indicating that this region may be functionally the most important for CRABP-I expression in the developing embryo.

Identification and characterization of the human type II collagen gene (COL2A1).

The gene contained in the human cosmid clone CosHcol1, previously designated an alpha 1(I) collagen-like gene, has now been identified. CosHcol1 hybridizes strongly to a single 5.9-kilobase mRNA species present only in tissue in which type II collagen is expressed. DNA sequence analysis shows that this clone is highly homologous to the chicken alpha 1(II) collagen gene. These data together suggest that CosHcol1 contains the human alpha 1(II) collagen gene COL2A1. The clone appears to contain the whole gene (30 kilobases in length) and will be extremely useful in the study of cartilage development and for identifying those inherited chondrodystrophies in which defects occur in this gene.

Beta-globin gene inactivation by DNA translocation in gamma beta-thalassaemia.

The beta-globin gene present on the deletion locus in a Dutch gamma beta-thalassaemic patient was found to be identical to the normal beta-globin gene with respect to DNA sequence and its transcription in HeLa cells. DNase I sensitivity and methylation experiments show that the affected beta-globin gene is present in an inactive configuration in vivo. This is the result of a translocation of a normally inactive locus next to the beta-globin gene on the affected chromosome, or the deletion of sequences which are normally required for the maintenance of the active state.

Regulated expression of the human beta-globin gene family in murine erythroleukaemia cells.

Chemically induced differentiation of cultured murine erythroleukaemia (MEL) cells results in a several hundred-fold increase in transcription of the adult mouse globin genes and thus serves as a model for gene activation during erythropoiesis. One approach to study gene regulation in this system has been to analyse the expression of foreign globin genes introduced into MEL cells. By introducing cosmid DNA containing the human adult(beta), fetal(gamma) and embryonic(epsilon)-globin genes, we have shown here that expression of the beta, but not the gamma or epsilon genes, is regulated during MEL differentiation. Regulated expression of the human beta-globin gene was observed when it was introduced either as part of the intact globin gene cluster or as an individual gene with 1.5 kilobases (kb) of 5' flanking DNA. Transcription from a herpes simplex virus (HSV) promoter adjacent to the thymidine kinase (tk) gene is also inducible in MEL cells.

Structural variations in the H-2 genes of AKR lymphomas.

K36.16 is an AKR H-2k thymoma which expresses an aberrant H-2Dd-like allospecificity, does not have a detectable amount of the H-2Kk syngeneic antigen and grows very easily in syngeneic mice. By DNA-mediated gene transfer experiments, we were able to obtain transformed clones which do express the H-2Kk molecules and are rejected by AKR mice. Southern hybridization was performed to assess whether any gross changes had occurred in the K36.16 H-2K locus or elsewhere in the MHC, which might explain the lack of H-2K expression and/or the presence of the aberrant H-2Dd-like allospecificity. Specific H-2 class I DNA probes were used to compare the K36.16 genomic DNA with normal AKR thymus DNA after digestion with a variety of restriction enzymes. After hybridization with the pH-2IIa probe a 2.8 kb 'Hind III' fragment was identified in the K36.16 genomic DNA which is absent from AKR DNA. The pH-2IIa probe detects the third, transmembrane and cytoplasmic domains of class I genes. Although these changes are indicative of MHC genome modifications it is not yet possible to link these specific Southern blot pattern variations with the phenotypic changes mentioned above.