Telomere shortening and cell cycle arrest in Trypanosoma brucei expressing human telomeric repeat factor TRF1

Trypanosoma brucei has telomeres composed of 15 kb tracts of TTAGGG repeats that end in 3' overhangs and form t-loops. This structure is also present in mammalian cells and is thought to reflect the presence of telomere-binding proteins. The human TTAGGG repeat-binding factor TRF1 binds to telomeres and regulates their length. We attempted to interfere with the normal function of trypanosome telomeres by expressing human TRF1 in T. brucei. TRF1 localized to telomeres in cultured procyclic (midgut-stage) trypanosomes with great fidelity, but not in bloodstream-stage trypanosomes. Procyclic trypanosomes expressing high levels of TRF1 for extended periods of time exhibited shortening and increased size heterogeneity of their telomeres and the cell cycle was arrested in G1-S. These effects were not detected in cells expressing a TRF1 mutant incapable of binding to TTAGGG repeats. We argue that TRF1 displaces putative endogenous trypanosome telomere-binding proteins, not yet identified, and affects telomeres in ways that reflect its role as a negative regulator of telomere length in human cells.     

Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains

The mouse CTLA-4 gene has been shown to code for an activated lymphocyte-associated sequence belonging to the Ig gene superfamily. We now report on the molecular cloning and study of the human corresponding gene isolated from a genomic library and designated Hu-CTLA-4. The Hu-CTLA-4 gene exists as a single copy per human haploid genome and maps to band q33 of chromosome 2. It comprises 3 exons notwithstanding the leader sequence. The first exon encodes a V-like domain of 116 amino acids, the second one a hydrophobic putative transmembrane region of 37 amino acids and the third one a 34 amino acid putative cytoplasmic domain. Whereas the overall homology between the human and murine CTLA-4 proteins is 76%, there is, remarkably, a complete identity of their cytoplasmic domains. This complete interspecies conservation comes in support of an important role for this domain in CTLA-4 function.     

Gene structure and chromosomal localization of the mouse homologue of rat OX40 protein

The OX40 protein is expressed only on activated rat CD4⁺ T blasts and is a member of a superfamily of cell surface molecules which includes CD40, CD30, CD95 (Fas), CD27, 4-1BB antigens and the receptors for tumor necrosis factor (TNF) and nerve growth factor (NGF). The proteins of this group are related to each other by having three to six repeats of a cysteine-rich sequence in their extracellular domains. Members of this family of receptors have also been shown to bind to ligands which are structurally related to TNF. The mouse homologue of the rat OX40 protein was cloned at the cDNA and genomic levels. The gene structure shows that there are several intron/exon borders shared between OX40 and CD27, CD40, TNF receptor type I, CD95 and 4-1BB genes. This group of genes is less closely related structurally to the gene structure of the NGF receptor. The gene encoding murine OX40 has been placed on mouse chromosome 4, in an area which contains the genes for TNF receptor type II and 4-1BB, and is syntenic with a region of human chromosome 1 which contains human TNF receptor type II, OX40, and CD30 genes.     

Cloning, expression analysis, and chromosomal localization of HIP1R, an isolog of huntingtin interacting protein (HIP1)

Huntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein. Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system. Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1. RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12. Received: May 18, 1998 / Accepted July 13, 1998     

Chromosomal localization ofCd1d genes in the mouse

Southern blot hybridization of DNA from Chinese hamster × mouse somatic cell hybrids was used to assign the mouseCd1d genes to chromosome 3. Analysis of the progeny of an intersubspecies backcross was used to position these genes near the gene for glucocerebrosidase,Gba.     

Characterization of genes encoding translation initiation factor eIF- 2gamma in mouse and human: sex chromosome localization, escape from X- inactivation and evolution

The Delta Sxrb interval of the mouse Y chromosome is critical for spermatogenesis and expression of the male-specific minor transplantation antigen H-Y. Several genes have been mapped to this interval and each has a homologue on the X chromosome. Four, Zfy1 , Zfy2 , Ube1y and Dffry , are expressed specifically in the testis and their X homologues are not transcribed from the inactive X chromosome. A further two, Smcy and Uty , are ubiquitously expressed and their X homologues escape X-inactivation. Here we report the identification of another gene from this region of the mouse Y chromosome. It encodes the highly conserved eukaryotic translation initiation factor eIF-2gamma. In the mouse this gene is ubiquitously expressed, has an X chromosome homologue which maps close to Dmd and escapes X-inactivation. The coding regions of the X and Y genes show 86% nucleotide identity and encode putative products with 98% amino acid identity. In humans, the eIF-2gamma structural gene is located on the X chromosome at Xp21 and this also escapes X-inactivation. However, there is no evidence of a Y copy of this gene in humans. We have identified autosomal retroposons of eIF-2gamma in both humans and mice and an additional retroposon on the X chromosome in some mouse strains. Ark blot analysis of eutherian and metatherian genomic DNA indicates that X-Y homologues are present in all species tested except simian primates and kangaroo and that retroposons are common to a wide range of mammals. These results shed light on the evolution of X-Y homologous genes.      

Genomic structure and tissue-specific expression of human and mouse genes encoding homologues of the major bovine seminal plasma proteins

Sperm capacitation is a maturation event that takes place in the female reproductive tract and is essential for fertilization. A family of phospholipid-binding proteins present in bovine seminal plasma (BSP proteins) binds the sperm membrane at ejaculation and promotes bovine sperm capacitation. Homologues of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid, suggesting that BSP proteins and their homologues are conserved among mammals. However, there have been no reports on BSP-homologous proteins in mice and humans to date. A search of the mouse and human genomes, using the nucleic acid sequences of BSP proteins, revealed the presence of three BSP-like sequences in the mouse genome, named mouse BSP Homologue 1 (mBSPH1), mBSPH2 and mBSPH3, and one sequence in the human genome (hBSPH1). Mouse epididymal expressed sequence tags corresponding to partial sequences of mBSPH1 and mBSPH2 were identified. The entire complementary DNA (cDNA) sequences of mBSPH1 and mBSPH2 from mouse epididymis and hBSPH1 from human epididymis were obtained by 5'-/3'-rapid amplification of cDNA ends (RACE) and encode predicted proteins containing two tandemly repeated fibronectin type II domains, which is the signature of the BSP family of proteins. Using RT-PCR, it was revealed that mBSPH1, mBSPH2 and hBSPH1 mRNA are expressed only in the epididymis. Expression of mBSPH3 was not detected in any tissue and probably represents a pseudogene. This work shows, for the first time, that BSP homologues are expressed in mouse and human and may be involved in sperm capacitation in these species.     

Decidual expression and localization of human surfactant protein SP-A and SP-D,and complement protein C1q

Surfactant proteins SP-A and SP-D, and complement protein C1q are soluble innate immune pattern recognizing molecules. SP-A, SP-D and C1q have an overall similar structure composed of an N-terminal triple-helical collagen region that is followed by a trimeric globular domain. While SP-A and SP-D belong to the collectin family (collagen containing lectin), C1q is the first recognition subcomponent of the classical pathway of the complement system. Recently, SP-A, SP-D and C1q have been considered to play important roles in early and late pregnancy. However, their expression in early human decidua has not been examined. Here, we investigated whether SP-A, SP-D and C1q are expressed within first trimester decidua in humans and their expression is associated with trophoblasts and decidual stromal cells. Decidual samples from women undergoing elective vaginal termination of pregnancy during first trimester were obtained from 25 subjects. Immunohistochemical studies using anti-human SP-A, anti-human SP-D and anti-human C1q antibodies were performed on decidual tissue sections along with anti-vimentin and cytokeratin-7 antibodies to identify stromal cells and trophoblasts. The expression was also examined by immunostaining and PCR using decidual and stromal cells. C1q expression was significantly higher when compared to SP-A and SP-D in the first trimester human decidua. Double immunostaining revealed that all stromal cells and trophoblasts expressed SP-A, SP-D and C1q, while only few invasive trophoblasts expressed C1q. Thus, expression of SP-A, SP-D and C1q in human decidua during first trimester suggests potential role of SP-A, SP-D and C1q during the early stages of pregnancy including implantation, trophoblast invasion and placental development.     

cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein

Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP). A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region. The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein. This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2. These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP. Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID). Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins. RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology. The human Fe65 mRNA was cloned from a fetal brain cDNA library. The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein. The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization.      

胶质瘤细胞ING1、人端粒酶逆转录酶及人端粒酶相关蛋白1基因表达的研究

目的 探讨胶质瘤细胞ING1、人端粒酶逆转录酶(hTERT)和人端粒酶相关蛋白1(hTP1)基因表达的意义。方法 用mRNA原位杂交及免疫组织化学染色方法观察了70例不同级别的人胶质瘤组织。结果 hTERT mRNA和蛋白的阳性表达率分别为88.6％和82.9％,hTP1 mRNA和蛋白的阳性表达率均为100％,这4种阳性肿瘤细胞的密度彼此间均呈正相关(r=0.758～0.882,P<0.0005),并均随肿瘤级别升高而相应增加(P<0.05～0.01)。ING1 mRNA和蛋白的阳性表达率分别为94.3％和88 6％,两种阳性肿瘤细胞密度间也呈正相关(r=0.831,P<0.0005),但两者均随肿瘤级别升高而相应减少(P<0.01),并均分别与hTERT mRNA和蛋白及hTP1 mRNA和蛋白的阳性肿瘤细胞密度呈负相关(r=-0.211～-0.384,P<0.05～0.001)。结论 胶质瘤细胞中hTERT和hTP1基因表达异常增加可能是抑制其ING1基因表达的重要因素,这3种基因的表达异常可能在胶质瘤发生及恶性进展过程中起重要作用。     

Two closely-related left-right asymmetrically expressed genes, lefty-1 and lefty-2: their distinct expression domains, chromosomal linkage and direct neuralizing activity in Xenopus embryos

Background:

Vertebrates have numerous lateral asymmetries in the position of their organs, but the molecular basis for the determination of left–right (L-R) asymmetries remains largely unknown. TGFβ-related genes such as lefty and nodal are L-R asymmetrically expressed in developing mouse embryos, and may be involved in L-R determination.

Results:

We have identified two highly conserved genes, lefty-1 and lefty-2, in the mouse genome. These two genes are tightly linked on mouse chromosome 1. lefty-1 and lefty-2 are both expressed in a L-R asymmetric fashion in mouse embryos. However, the major expression domains of the two genes are different: lefty-1 expression is predominantly confied to the left side of ventral neural tube, whereas lefty-2 is strongly expressed in the lateral plate mesoderm on the left side. In embryos homozygous for the iv and inv mutation, which cause situs inversus, the expression sites of both genes are affected, either reversed or bilaterally, indicating that lefty-1 and lefty-2 are downstream of iv and inv. Although Lefty-1 and Lefty-2 prepro-proteins are not readily processed in cultured cells, BMP2-Lefty chimeric proteins can be processed to a secreted form. We have examined the activities of Lefty-1 and Lefty-2 in Xenopus embryos. In animal cap explants, Lefty-1 and Lefty-2 induce neural cells in the absence of mesoderm induction. The direct neuralizing activities of Lefty-1 and Lefty-2 thus seem remarkably similar to those of BMP antagonists such as noggin and chordin, suggesting that the action of Lefty-1 and Lefty-2 may be to locally antagonize BMP (bone morphogenic protein)-mediated signals in tissues positioned on the left side of the mouse embryos.

Conclusion:

There are two lefty genes in mice (lefty-1 and lefty-2), both of which are expressed in a L-R asymmetric fashion and are downstream of iv and inv. Lefty-1 and Lefty-2 possess direct neuralizing activity in Xenopus embryos, resembling the activities of BMP antagonists.