Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

Background

Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.

Results

We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.

Conclusion

A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.     

The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae

The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a- factor receptor, Ste3p. Mutation of this sequence prevented pheromone- stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast.     

The yeast gene MSH3 defines a new class of eukaryotic MutS homologues

We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse. MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS. MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation. However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes. Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors. MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair. We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.     

The RAG cell line defines a new complementation group of MHC class II deficiency

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN- ofDMA, DMB, and theinvariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designatedF16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding humanCIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.     

Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI.     

The yeast gene MSH3 defines a new class of eukaryotic MutS homologues

We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse. MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS. MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation. However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes. Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors. MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair. We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.     

EST analysis in barley defines a unigene set comprising 4,000 genes

We report the generation of 13,109 EST (Expressed Sequence Tag) sequences from barley as a first step towards the generation of a unigene set for this organism. Sequences were generated from three libraries encompassing 7,568 cDNA clones. Comparisons to nucleic acid and protein sequence databases enabled the assignment of putative functions to the mRNAs. The results of the searches against protein databases were parsed and built into a regularly updated database, available over the World Wide Web. The Stack_Pack clustering system has been applied to survey the level of redundancy, which was calculated to amount to 69%, thus we identified 4,000 different barley genes. To prove the usability of the results of the clustering process for further experiments, we subjected alignments with sequences similar to elongation factor 1 alpha to additional analysis. These sequences represented the largest group with identical putative functions (228 members) and clustering based on the analysis of 3′ sequences subdivided the group into five different assemblies. Alignments of the consensus sequences facilitated the development of PCR assays suitable for genetic mapping of four of the different gene-family members, which reside on chromosomes 2H, 4H and 5H, thus demonstrating the suitability of the cluster-results as a basis for in-depth analyses of barley gene families. Received: 15 March 2001 / Accepted: 18 April 2001     

The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.     

The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.

Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3'' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of PUM to the NRE is thought to target hb(mat) mRNA for repression. Here, we show the RNA-binding domain of PUM to be an evolutionarily conserved, 334-amino acid region at the carboxy-terminus of the approximately 158-kDa PUM protein. This contiguous region of PUM retains the RNA-binding specificity of full-length PUM protein. Proteins with sequences homologous to the PUM RNA-binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the PUM RNA-binding domain in other far-flung species suggests that the domain is an ancient protein motif, and we show that conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila PUM.     

Pod-2, along with pod-1, defines a new class of genes required for polarity in the early Caenorhabditis elegans embryo

The asymmetric division of the one-cell Caenorhabditis elegans zygote gives rise to two cells of different size and fate, thereby establishing the animal's anterior--posterior (a-p) axis. Through genetics, a number of genes required for this polarity have been characterized, but many components remain unidentified. Recently, our laboratory discovered a mutation in the pod-1 gene (for polarity and osmotic defective) that uniquely perturbed polarity and osmotic protection. Here, we describe a new C. elegans polarity gene identified during screens for conditional embryonic lethals. Embryos in which this gene has been mutated show a loss of physical and developmental asymmetries in the one-cell embryo, including the mislocalization of PAR and POD-1 proteins required for early polarity. Furthermore, mutant embryos are osmotically sensitive, allowing us to designate this gene pod-2. Thus, pod-2, along with pod-1, defines a new class of C. elegans polarity genes. Genetic analyses indicate that pod-2 functions in the same pathway as pod-1. Temperature-shift studies indicate that pod-2 is required during oogenesis, indicating that aspects of embryonic polarization may precede fertilization. pod-2 mutant embryos also exhibit a unique germline inheritance defect in which germline identity localizes to the wrong spot in the one-cell embryo and is therefore inherited by the wrong cell at the four-cell stage. Our data suggest that pod-2 may be required to properly position an a-p polarity cue.     

The spasmodic peptide defines a new conotoxin superfamily

We purified and characterized a peptide from the venom of Conus textile that makes normal mice assume the phenotype of a well-known mutant, the spasmodic mouse. This "spasmodic" peptide has 27 amino acids, including two gamma-carboxyglutamate (Gla) residues. A cDNA clone encoding the precursor for the peptide was identified; a gamma-carboxylation recognition signal sequence (gamma-CRS) is present in the -1 --> -20 region of the peptide precursor. Both the gamma-CRS and the position of the Gla residues in the mature toxin are notably different from other Gla-containing conopeptides. The spasmodic peptide has a novel disulfide framework and distinct signal sequence which together define a new P-superfamily of conopeptides. A cDNA encoding another member of the P-superfamily was identified from a different species, Conus gloriamaris.     

New inducers revealed by the promoter sequence analysis of two interferon-activated human genes

In order to investigate the molecular basis of the regulation of interferon-inducible genes, we isolated the promoter region of two such genes coding for the (2'-5')oligo(adenylate) synthetase and a 56-kDa protein (IFI-56K). The regions surrounding the cap site were sequenced and compared with the sequences of vertebrate and viral DNA present in the Genbank data bank. Small DNA segments were found in both genes which are homologous to part of the promoter region of other genes, such as those of interferon-beta, tumor necrosis factor beta, interleukin-2 and its receptor. Since these homologies were found located in functionally important regions of these genes, we tested whether their inducers also enhance the (2'-5')oligo(adenylate) synthetase and IFI-56K gene expression. We found that poly(rI).poly(rC) and interleukin-1, activators of the interferon-beta gene and of T lymphocytes respectively, are both able to enhance IFI-56K mRNA accumulation in all cell lines tested. Cycloheximide even superinduces this gene when added together with poly(rI).poly(rC) and interleukin-1 (but not when added with interferon). We showed that these inductions are direct and not mediated by interferon produced by cells in response to poly(rI).poly(rC) or interleukin-1. The promoter sequence analyses have thus led to the discovery of unexpected inducers, i.e. an interferon inducer such as poly(rI).poly(rC) is also able to directly induce a gene that is under the control of interferon.