A CDNA selection approach to isolate Y-linked genes expressed in testis

Statistical analysis has revealed that 2-7% of couples remain childless at the end of their reproductive life. In about half of these cases, the problem lies with the male partner. It is expected that genes expressed in the testis, and involved in spermatogenesis, will occur on the Y-chromosome. This view has been confirmed with the isolation of the Y-linked RBMY, TSPY and DAZ genes, deletions of which appear to be present in cases of oligo- and azoospermia. In an effort to identify further Y-linked, testis-expressed genes, a cDNA selection library that was made by selecting testis cDNA with 1,000 Y- linked cosmid clones, was screened for Y-specific cDNAs. Screening with vector sequences and Y-linked repeat sequences, led to the elimination of more than 80% of the clones. Of the remaining 731 potential cDNA clones, sequencing revealed 79 clones with homology to several sequences in the database, including Y-linked and testis related sequences. A number of sequences isolated from the cDNA selection library appeared to represent novel members of two gene families and they were further investigated. These studies revealed that the TTY2 gene, published by Lahn and Page (1997), is member of a large Y-linked multicopy gene family with an estimated number of 26 copies. Two members of this gene family, termed ml 13d 10 and ml22a3 TTY2-like genes, were further investigated, as representatives of the TTY2 gene family. One more cDNA clone, termed 22d8, showed homology with several ESTs of unknown function and a 3.4kb cDNA that was recently released in the database (Feb. 2000) from a testis cDNA library. Investigation of this potential gene, revealed that either the whole, or part of it, is present in several chromosomal locations, including the Y chromosome and that it is ubiquitously expressed in many tissues, including testis

Applications of Molecular diagnosis in soft tissue tumors

This article concerns the contribution of new technological advances in the field of molecular genetics and their application for a more precise classification of soft tissue tumors. Molecular techniques offer new information about the heterogeinity and complexity of tumors, since we have at present a plethora of information related to genes and gene regulators that contribute to the complex interactions between environment and genetic background. The use of molecular diagnosis offers a more reliable classification system and can utilize even minute tissue samples. In addition, it can help improve new therapeutic ways, including gene therapy

A Splicing Mutation in the Novel Mitochondrial Protein DNAJC11 Causes Motor Neuron Pathology Associated with Cristae Disorganization, and Lymphoid Abnormalities in Mice

Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases

TTY2: A Multicopy Y-Linked Gene Family

Genes involved in human male sex determination and spermatogenesis are likely to be located on the Y chromosome. In an effort to identify Y-linked, testis-expressed genes, a cDNA selection library was generated by selecting testis cDNA with Y-cosmid clones. Resultant clones containing repetitive or vector material were eliminated, and 79 of the remaining clones were sequenced. Nineteen cDNAs showed homology with the TTY2 gene, and indicated that TTY2 is part of a large gene family. Screening of a panel of Y-linked cosmids revealed that the TTY2 gene family includes at least 26 members organized in 14 subfamilies. Further investigation revealed that TTY2 genes are arranged in tandemly arrayed clusters on both arms of the Y chromosome, and each gene comprises a series of tandemly arranged repeats. RT-PCR studies for two of these genes revealed that they are expressed in adult and fetal testis, as well as in the adult kidney. None of the genes investigated in detail contain an open reading frame. We conclude that the TTY2 gene family is composed of multiple copies, some of which may function as noncoding RNA transcripts and some may be pseudogenes. [The sequence data described in this paper for TTY2L12A and TTY2L2A have been submitted to the EMBL data library, under accession nos. AJ297963 and AJ297964.

Mitochondrial and submitochondrial localization of DNAJC11.

<p>(A) Representative Western blot analysis on total protein RIPA extracts (T), on cytosolic (C) and mitochondrial (M) fractions of cerebrum and liver tissue from WT mice. The hu<i>DnaJC11</i> transgenic (Tg) samples served as positive controls. Prohibitin served as a mitochondrial specific marker and GAPDH as a cytoplasmic marker. (B) Fluorescence microscopy of HeLa cells transfected with a C-terminally FLAG-tagged huDNAJC11 cDNA of the 63 kDa isoform (green channel) and stained with the mitochondrial specific dye MitoTracker Orange (red channel). Scalebar, 100 µm. (C) Proteinase K protection assay on isolated mitochondria from HeLa cells. 50 µg of mitochondria were either subjected to swelling in the hypotonic buffer (SW, +) or were incubated in the isotonic buffer (SW, -) and then were treated with proteinase K (PK, +) or not (PK, -). A buffer containing 1% TritonX-100 (Tr) was used to solubilize all mitochondrial proteins and render them accessible to proteinase K. Samples were then analyzed by SDS-PAGE and Western blot with the indicated antibodies. Metaxin 1 is an outer membrane protein, mitofilin is an intermembrane space exposed inner membrane protein and Hsp60 is matrix localized. Numbers indicate molecular mass of protein marker in kDa. (D) Sodium carbonate extraction of isolated mitochondria from HeLa cells. Mitochondria were extracted under the two indicated pH conditions and the membranes were collected by ultracentrifugation. Pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blot and probed with the indicated antibodies. Tim23 is an integral inner membrane protein and ICDH is a soluble matrix protein. Arrows indicate the 63 kDa isoform and the putative ∼57 kDa isoform. Tim23, Translocase of inner mitochondrial membrane 23 homolog; ICDH, isocitrate dehydrogenase.</p

Complete rescue of the <i>DnaJC11^spc/spc</i> phenotype through expression of the human <i>DnaJC11</i> gene.

<p>(A) Schematic representation of the human BAC clone fragment that was used for the generation of Tg<i>huDnaJC11</i> mice. Genes and their orientation are indicated as well as NotI sites that were used for digestion. Horizontal line and number below represent the fragment length. (B) Copy number determination, by qPCR, of three transgenic lines, TgF843, TgF867, and TgF869 (n = 5-9 per group) using a primer pair common for both mouse and human <i>DnaJC11</i> genes. WT mice were considered to carry 2 copies of <i>DnaJC11</i>. (C) Body weight and (D) grip strength (normalized to body weight) curves for the indicated genotypes. All mice used were sex matched littermates, (n = 8). (E) Rescue of the thymic hypoplasia shown as total thymic cellularity, (n = 3). (F) Restoration of thymic subpopulations distribution in rescued mice (n = 3) as determined by flow cytometry after staining with antibodies against CD4 and CD8. Statistical analysis between controls and rescued (Tg/<i>DnaJC11^spc/spc</i>) mice is indicated. DP, double positive; DN, Double Negative. (G) Restoration of splenic subpopulations distribution in rescued mice. B cells and myeloid cells were defined as the ones positive for markers B220 and CD11b respectively, (n = 3). (H) Restoration of the leucopenia phenotype and the increased red blood cell phenotype in rescued mice (n = 3). Data represent means ± SE.</p

Lymphoid and blood abnormalities in <i>spc</i> mice.

<p>(A) Representative thymi dissected from <i>spc/spc</i> and WT (+/+) littermate mice at 4 weeks of age. Scalebar, 5mm. (B) Representative H/E stained thymic sections from <i>spc/spc</i> and +/+ littermate mice (n = 4). Scalebar, 400 µm. (C) Total thymus cellularity in <i>spc/spc</i> mice and control littermates, (n = 14 per group). (D) Percentage of thymic subpopulations in <i>spc/spc</i> mice and control littermates as determined by flow cytometry after staining of thymocytes with antibodies against CD4 and CD8. Data represent means ± SE from four independent experiments, (n = 13 per group). (E) Percentages of CD4^-CD8^- double negative (DN) subpopulations as determined by flow cytometry after staining of thymocytes with antibodies against CD25 and CD44, in <i>spc/spc</i> and control littermates, (n = 6 per group). (F) Representative spleens dissected from <i>spc/spc</i> and +/+ littermate mice at 4 weeks of age. Scalebar, 5 mm. (G) Total spleen cellularity of <i>spc/spc</i> mice and control littermates, (n = 14 per group). (H) Percentage of splenic subpopulations in <i>spc/spc</i> mice and control littermates as determined by flow cytometry using antibodies against CD4, CD8, B220 (B cells), Gr1 and CD11b (Myeloid). Data represent means ± SE from four independent experiments, (n = 10 per group). (I) Peripheral blood counts of <i>spc/spc</i> mice and control littermates, (n = 7 per group). Controls presented in bar graphs are healthy littermates (+/+ and <i>+/spc</i>).</p

Total Blood Counts in <i>spc/spc</i> mice.

<p>WBCs: White blood cells, RBCs: Red blood cells, RDW: Red cell distribution width MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, PCT: Plateletcrit, MPV: Mean platelet volume, PDW: Platelet distribution width, MXD: Mixed cell count. Data are expressed as mean ± SE, (n = 7 per group). Control mice (+/+ and <i>spc</i>/+) were sex matched littermates. Student's t test was performed for statistical analysis. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Expression analysis of mutant muDNAJC11 and biochemical interaction of huDNAJC11 with MICOS members.

<p>(A) Western blot analysis of fractionated brain tissue from <i>DnaJC11^spc/spc</i> (<i>spc/spc</i>) mice and WT (+/+) littermates showing the loss of muDNAJC11 protein in <i>DnaJC11^spc/spc</i> tissue. Prohibitin is a mitochondrial specific marker and GAPDH a cytoplasmic marker. (B) Equal amounts of isolated mitochondria from the indicated mouse tissues were analyzed by Western blot and probed for known members of the MICOS complex. Glucose related protein 75 (GRP75) was used as a loading control. (C) <i>dnajc11kd</i>-3, (D) <i>sam50kd</i>-2 or (E) <i>mflkd</i>-2 cells were grown in the absence (-Dox) or presence (+Dox) of doxycycline for 7 or 14 days, mitochondria were isolated, and 25 or 50 µg of protein were analyzed by SDS-PAGE and probed for the indicated proteins. SDHA, the component of the respiratory complex II, was used as a loading control. CHCHD3 and 6, coiled-coil-helix-coiled-coil-helix domain containing protein 3 and 6; SAM50, Sorting and assembly machinery 50; SDHA, Succinate Dehydrogenase subunit A. (F) Mitochondria from non-induced and induced <i>sam50kd-2</i> knockdown cells after 7 days of induction with doxycyclin (Dox) were isolated and incubated with the radiolabeled mitofilin and DNAJC11 (the longest isoform) for the indicated time periods. Samples were analyzed by BN-PAGE and autoradiography. The panel on the right hand side shows the control of the knockdown, where 50 µg of mitochondria from –Dox and +Dox samples were analyzed by SDS-PAGE and Western blot using antibodies against Sam50 and SDHA.</p