Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

The behavior of SATB1, a MAR-binding protein, in response to apoptosis stimulation

As a MAR-binding protein, SATB1 regulates genes by folding chromatin into a loop domain. Apoptosis is known to be accompanied by a collapse of nuclear architecture and cleavage of condensing chromatin into oligonucleosomal fragments. To further understand the functional role of MAR-binding proteins during apoptosis we investigated the relationship of the behavior of SATB1 and the collapse of nuclear architecture in Jurkat cells with immunostaining and Western blot analysis. We demonstrated that SATB1 formed special three-dimensional network distributions during early apoptosis. The distribution change of SATB1 was associated with cleavage of the protein and accompanied by the nuclear architecture collapse. Cleavage of SATB1 was mediated by caspase-3 and was apoptosis specific. Our observations further support the notion that early proteolysis of MAR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.     

Identification of New Regulatory Sequences Far Upstream of the Mouse Monocyte Chemoattractant Protein-1 Gene

We systematically searched for sequences influencing the expression of the mouse monocyte chemoattractant protein-1 (MCP-1) gene (Scya2) by mapping DNase I hypersensitive sites (HS) in the chromatin of mesangial cells in a 40-kb interval around the gene. We found nine HS located between -24 kb and +12.7 kb. Three HS coincided with previously known regulatory sequences (HS-2.4, HS-1.0, and HS-0.2). We tested two of the previously unknown HS located far upstream of Scya2 (HS-19.4 and HS-16.3) in transfection experiments using luciferase reporter constructs and mouse mesangial cells as recipients. In transient transfections, both HS had a moderate effect on basal promoter activity as well as promoter activity stimulated by tumor necrosis factor-alpha. In stable transfection experiments, we found much higher activity. A DNA fragment containing HS-19.4 and HS-16.3 caused a considerable increase in the number of stably integrated luciferase copies. We determined the nucleotide sequence of the 5' flanking region to -28.6 kb. Computer-assisted sequence analysis did not yield evidence of an additional gene. These HS are located within the 5' flanking region of a gene cluster consisting of Scya2 (MCP-1), Scya7 (MCP-3), Scya11 (eotaxin), Scya12 (MCP-5), and Scya8 (MCP-2). This report represents the first comprehensive chromatin analysis of the mouse MCP-1 locus leading to the identification of a complex regulatory region located far upstream of Scya2.     

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma

Using differential display PCR, we identified a novel gene upregulated in renal cell carcinoma. Characterization of the full-length cDNA and gene revealed that the encoded protein is a human homologue of the Drosophila melanogaster Tweety protein, and so we have termed the novel protein TTYH2. The orthologous mouse cDNA was also identified and the predicted mouse protein is 81% identical to the human protein. The encoded human TTYH2 protein is 534 amino acids and, like the other members of the tweety-related protein family, is a putative cell surface protein with five transmembrane regions. TTYH2 is located at 17q24; it is expressed most highly in brain and testis and at lower levels in heart, ovary, spleen, and peripheral blood leukocytes. Expression of this gene is upregulated in 13 of 16 (81%) renal cell carcinoma samples examined. In addition to a putative role in brain and testis, the over-expression of TTYH2 in renal cell carcinoma suggests that it may have an important role in kidney tumorigenesis.     

The pattern of gene expression in mouse Gr-1(+) myeloid progenitor cells

To understand the pattern of gene expression in mouse myeloid progenitor cells, we carried out a genome-wide analysis of gene expression in mouse bone marrow Gr-1(+) cells using SAGE and GLGI techniques. We identified 22,033 unique SAGE tags with quantitative information from 73,869 collected SAGE tags. Among these unique tags, 64% match known sequences, including many genes important for myeloid differentiation, and 36% have no matches to known sequences and are likely to represent novel genes. We compared the expression of mouse Gr-1(+) and human CD15(+) myeloid progenitor cells and showed that the pattern of gene expression of these two cell populations had some similarities. We also compared the expression of mouse Gr-1(+) myeloid progenitor cells with that of mouse brain tissue and found a highly tissue-specific manner of gene expression in these two samples. Our data provide a basis for studying altered gene expression in myeloid disorders using mouse models.     

Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specific for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.     

Several cardiomyopathy causing mutations on tropomyosin either destabilize the active state of actomyosin or alter the binding properties of tropomyosin

We examined the cardiomyopathy-causing tropomyosin mutations E180G, D175N, and V95A to determine their effects on actomyosin regulation. V95A reduced the ATPase rate when filaments were saturated with regulatory proteins both in the presence and absence of calcium, indicating either a stabilization of the inactive state or an inability to fully populate the active state. Effects of E180G and D175N were more complex. These two mutations increased ATPase rates at sub-saturating concentrations of troponin and tropomyosin as compared to wild type tropomyosin. At higher concentrations of regulatory proteins, ATPase rates became similar to wild type. Normal activation was achieved with the tight-binding myosin analog N-ethylmaleimide-S1, at saturating regulatory protein concentrations. These results suggest that the E180G and D175N mutations reduce the affinity of tropomyosin for actin and also destabilize troponin binding to the actin thin filaments.     

Identification of a novel protein, PriB, in Klebsiella pneumoniae

PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L₄₅, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed.     

The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h

The tomato AGC protein kinase Adi3 is known to function as a suppressor of PCD and silencing of Adi3 leads to spontaneous cell death on leaves and stems. In an effort to isolate Adi3 interacting proteins, a yeast two-hybrid screen was carried out and identified the autophagy protein Atg8h as an Adi3 interactor. This interaction occurred independent of the kinase activity status of Adi3. Silencing of genes involved in autophagy is known to eliminate the restriction of pathogen-induced PCD to a few cells and leads to run away PCD. Cosilencing Adi3 with several autophagy genes lead to the same run away cell death suggesting Adi3 may be involved in autophagic regulation of PCD.     

Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1

The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.     

Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region

UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F⁶¹⁹ in the motif stacks partly over cis-Pro⁶²⁰ in one conformation, whereas it is swung out from cis-P⁶²⁰, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCP-binding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP.     

RNA polymerase II degradation in response to rapamycin is not mediated through ubiquitylation

In Saccharomyces cerevisiae, the immunosuppressor rapamycin engenders the degradation of excessive RNA polymerase II leading to growth arrest but the regulation of this process is not known yet. Here, we show that this mechanism is dependent on the peptidyl prolyl cis/trans isomerase Rrd1. Strikingly this degradation is independent of RNA polymerase II polyubiquitylation and does not require the elongation factor Elc1. Our data reveal that there are at least two alternative pathways to degrade RNA polymerase II that depend on different type of stresses.