ABSTRACT
PDZ motifs are modular protein-protein interaction domains, consisting of 80-120 amino acid residues, whose function appears to be the direction of intracellular proteins to multiprotein complexes. In skeletal muscle, there are a few known PDZ-domain proteins, which include neuronal nitric oxide synthase and syntrophin, both of which are components of the dystrophin complex, and actinin-associated LIM protein, which binds to the spectrin-like repeats of alpha-actinin-2. Here, we report the identification and characterization of a new skeletal muscle protein containing a PDZ domain that binds to the COOH-terminal region of alpha-actinin-2. This novel 31-kD protein is specifically expressed in heart and skeletal muscle. Using antibodies produced to a fragment of the protein, we can show its location in the sarcomere at the level of the Z-band by immunoelectron microscopy. At least two proteins, 32 kD and 78 kD, can be detected by Western blot analysis of both heart and skeletal muscle, suggesting the existence of alternative forms of the protein. In fact, several forms were found that appear to be the result of alternative splicing. The transcript coding for this Z-band alternatively spliced PDZ motif (ZASP) protein maps on chromosome 10q22.3-10q23.2, near the locus for infantile-onset spinocerebellar ataxia.
Subject(s)
Alternative Splicing , Carrier Proteins/genetics , Homeodomain Proteins , Muscle Proteins/genetics , Sarcomeres/metabolism , Actinin/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Chromosomes, Human, Pair 10/genetics , Cloning, Molecular , Fluorescent Antibody Technique , Heart/embryology , Humans , LIM Domain Proteins , Mice , Microscopy, Immunoelectron , Molecular Sequence Data , Molecular Weight , Muscle Proteins/chemistry , Muscle Proteins/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscle, Skeletal/ultrastructure , Myocardium/metabolism , Myocardium/ultrastructure , Organ Specificity , Precipitin Tests , Protein Binding , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sarcomeres/ultrastructure , Yeasts/geneticsABSTRACT
We describe the structure, genomic organization, and some transcription features of a human brain-specific gene previously localized to the genomic region involved in temporal lobe epilepsy and spastic paraplegia on chromosome 10q24. The gene, which consists of six exons disseminated over 16 kb of genomic DNA, is highly homologous to the porcine tmp83.5 gene and encodes a putative transmembrane protein of 141 amino acids. Unlike its porcine homolog, from which two mRNAs with different 5'-sequences are transcribed, the human gene apparently encodes three mRNA species with 3'-untranslated regions of different sizes. Mutation analysis of its coding sequence in families affected with temporal lobe epilepsy or spastic paraplegia linked to 10q24 do not support the involvement of this gene in either diseases.
Subject(s)
Brain/metabolism , Chromosomes, Human, Pair 10/genetics , Epilepsy, Temporal Lobe/genetics , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Paraplegia/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , DNA, Complementary/chemistry , DNA, Complementary/genetics , Exons , Gene Expression , Genes/genetics , Humans , Introns , Molecular Sequence Data , Mutation , Myelin Proteins , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , SwineSubject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Genes, Insect , Insect Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Cell Cycle Proteins , Circadian Rhythm/genetics , Evolution, Molecular , Gene Duplication , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
PD1 is a novel protein particularly expressed at the testicular level. The relative cDNA sequences were cloned from human and rat testis libraries revealing an open reading frame for a protein of 520 and 511 amino acids respectively. The human PD1 amino acid sequence shows 85% identity with rat sequence suggesting that PD1 gene has been highly conserved during mammalian evolution. Immunohistochemical analysis showed that this protein is detected in the tubular compartment of the testis and, in particular, in the cytoplasm of the Sertoli cells. PD1 expression is not constitutive but seems to be under the influence of neighboring spermatogenic cells as demonstrated by its reduction in hypospermatogenesis with respect to normal spermatogenesis and a further reduction in Sertoli cell-only syndrome. During testicular development in the rat (from 2 to 45 days of age) the PD1 mRNA level became detectable at 14 days and then increased steadily with an advancement of age. These findings suggest that PD1 may play a role in the regulation of spermatogenesis and may be a potential candidate gene for defects of male fertility.
Subject(s)
Proteins/genetics , Spermatogenesis/genetics , Amino Acid Sequence , Animals , Humans , Male , Molecular Sequence Data , Proteins/chemistry , Proteins/physiology , RNA, Messenger/analysis , Rats , Sequence Homology , Testis/chemistryABSTRACT
We have cloned and sequenced a cDNA from a human adult skeletal muscle cDNA library, encoding for a novel isoform of alpha-tubulin (tuba8) that is preferentially expressed in heart, skeletal muscle, and testis. A genomic DNA sequence from the chromosomal region 22q11 allowed us to determine the complete structure of the TUBA8 gene that mirrors the canonical exon/intron organization of the vertebrate alpha-tubulin genes. We also cloned and sequenced the cDNA of its murine homologue (MMU-TUBA8). The latter encodes for a protein that differs from its human counterpart in only three amino acids, revealing an extreme rate of conservation that is even extended to both the 3' and 5' UTRs of the mRNAs. Sequence comparison of these novel isoforms with other known alpha tubulins shows that tuba8 is the most divergent member of the mammalian alpha-tubulin family. The sequence peculiarity of the human and murine tuba8 strongly suggests that they might have functional significance and, according to the multi-tubulin hypothesis, that they might play specific functional roles in the cell cytoskeleton.
Subject(s)
Chromosomes, Human, Pair 22 , Muscle, Skeletal/metabolism , Tubulin/genetics , Adult , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Conserved Sequence , Humans , Mammals , Mice , Molecular Sequence Data , Phylogeny , Protein Isoforms/chemistry , Protein Isoforms/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Tubulin/chemistryABSTRACT
A novel human cDNA sequence has been isolated from human testis cDNA library. This sequence, named PD1, reveals an open reading frame encoding a protein of 520 amino acids. A partial sequence similarity has been found with the RBM gene involved in the regulation of human spermatogenesis. Northern blot analysis for PD1 mRNA from several human tissues demonstrated two distinct transcripts of 2.7 (more abundant) and 4.0 kb and revealed that PD1 is expressed in testis and to a lesser extent also in spleen, thymus, and prostate. Immunohistochemical analysis of human testis showed that this protein is detected in the cytoplasm of Sertoli cells. Antibodies against a rhPD1 fragment were used for Western blot analysis, which confirmed the presence of a 60-kDa molecule in crude extract of human testicular cells obtained from fine-needle aspiration and showed different patterns in various testiculopathies, suggesting a role for such gene in human spermatogenesis.
Subject(s)
Proteins/genetics , Testis/chemistry , Testis/pathology , Amino Acid Sequence , Base Sequence , Blotting, Northern , Blotting, Southern , Cloning, Molecular , DNA, Complementary/genetics , Humans , Male , Molecular Sequence Data , Nuclear Proteins , Proteins/isolation & purification , RNA-Binding Proteins/genetics , Seminiferous Tubules/chemistry , Sequence Homology, Amino Acid , Sertoli Cells/chemistry , Sperm Count , Spermatogenesis , Tissue DistributionABSTRACT
A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared with D123, three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in alpha-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified.
Subject(s)
Cell Cycle Proteins , Proteins/genetics , Amino Acid Sequence , Amino Acids/genetics , Animals , Base Sequence , Blotting, Northern , Blotting, Western , Cell Line , Cell Line, Transformed , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Gene Expression/genetics , Humans , Immunohistochemistry , Male , Molecular Sequence Data , Protein Processing, Post-Translational , Proteins/analysis , RNA, Messenger/metabolism , Rabbits , Rats , Sequence Alignment , Sequence Analysis, DNA , Statistics as Topic , Testis/chemistry , Tissue Distribution , Tumor Cells, CulturedABSTRACT
Over-production of gelatinase A (MMP-2) or under-production of its inhibitor (TIMP-2) may result in the matrix degradation crucial for metastasis, and early evaluation of their expression in primary tumor would offer important prognostic informations. RT-PCR amplicons of MMP-2 and TIMP-2 mRNA from tissue biopsies of 13 breast carcinomas and one fibrocystic mastopathy were quantitated. In comparison with their normal-tissue counterparts, their expression trends were not uniform: in some cases MMP-2 increased in the tumor without changes in TIMP-2, in others TIMP-2 expression also increased, although to a lesser extent than MMP-2; only in 2 cases was it slightly lower in the tumor tissue. Nevertheless, clearer insights were gained from the comparison of the ratio (R) between MMP-2tumor/normal and TIMP-2tumor/normal: as in the fibrocystic mastopathy, the R in carcinomas without lymph-node involvement (LN-) was usually lower than I in most cases. In contrast, in 5 out of 6 patients with lymph-node metastasis (LN+), the ratio ranged between 2 and 4. While the R magnitude was not related to the frequency of positive lymph nodes out of the total analyzed, nor to relapse status at follow-up (all relapse-free), the clear-cut difference between the LN- and LN+ groups was statistically significant. Results suggest that evaluation of MMP-2/TIMP-2 mRNA balance may constitute an early prognostic approach, which may also be more reliable concerning cancer aggressiveness as compared with the MMP-2 alone, and that boosting TIMP-2 expression may be a therapeutic strategy to prevent metastasis.