A thrombospondin homologue in Drosophila melanogaster: cDNA and protein structure.

The cDNA of a thrombospondin homologue in Drosophila melanogaster (DTSP) has been sequenced, and structural features of the translated protein analyzed. Thrombospondin proteins had previously been identified only in vertebrates, including human and mouse. Comparison with the genomic sequence revealed that the DTSP gene is divided into 13 exons; The translation initiation site is in exon 2. The transcription start site was analyzed using a PCR procedure, and the longest transcripts were found to initiate about 300 bp 5' of the predicted start site. The open reading frame of the DTSP cDNA encodes a protein that has 1060 amino acid residues. The polypeptide is composed of domains or repeats characteristic of the TSP3/TSP4/COMP subfamily of thrombospondin proteins: Amino-terminal domain, four Type II repeats, seven Type III repeats, carboxyl-terminal domain. The protein is highly acidic, particularly in the region of Type III repeats, with an Asp + Glu content of 15.8%. A signal peptide was detected at the N-terminus, which indicates that DTSP, like other TSPs, functions as an extracellular protein. Ten Asn residues were identified as potential glycosylation sites. Alignment of the amino acid sequences of the Drosophila TSP with human TSP1-TSP4 and COMP demonstrated a high degree of homology between the four Type II repeats, seven Type III repeats, and C-terminal domain.

Relationship of transcription of Drosophila melanogaster gene CG11327 and the gene for a thrombospondin homologue (DTSP).

Because of the close proximity of D. melanogaster gene CG11327 and the gene for a thrombospondin homologue (DTSP), the relationship between transcription of the two genes has been investigated. As part of this study, the cDNA sequence of gene CG11327 was determined, and structural features of the encoded protein were characterized. The CG11327 open reading frame specifies a polypeptide of 475 amino acid residues, molecular weight 53,666. The highly acidic protein has an aspartic acid + glutamic acid content of 23.2%. Weak similarities to other proteins suggest that it may function as a transcription factor or structural protein of the cell cycle. The existence of overlapping transcripts was found to be a significant feature of transcription of gene CG11327 and the DTSP gene. The 3' end of the DTSP gene overlaps the 5' end of gene CG11327 by at least 177bp, and the region of overlap includes exon 13 and the 3' UTR of the DTSP gene.

The human thrombospondin 3 gene: analysis of transcription initiation and an alternatively spliced transcript.

Thrombospondin 3 (TSP3) is a member of a family of modular, extracellular proteins that have been implicated in a diverse number of important biological processes. To contribute to an understanding of the precise roles of human TSP3, aspects of TSP3 gene transcription have been investigated. The TSP3 gene (THBS3) shares a promoter/intergenic region of 1.4 kb with the divergently transcribed metaxin gene, and the existence of TSP3 transcription initiation sites in the TSP3/metaxin intergenic region was investigated by a PCR procedure. Transcripts were detected which initiate in the intergenic region, up to several hundred bases upstream from the major transcription start site. An alternatively spliced transcript of TSP3 was also detected by the PCR procedure. This includes a new exon, exon A', which replaces exon A. Exon A' is located in the TSP3/metaxin intergenic region, 1 kb 5' of exon A. In addition, transcripts of metaxin were found with extended 5' ends; these overlap the 5' end of the TSP3 alternative transcript. The complexities of TSP3 transcription initiation revealed by this study could contribute to the tissue-specific expression and diverse functions of TSP3.

Detection of exon skipping and retained introns in transcription of the human thrombospondin 2 gene.

Alternative transcripts of the gene for human thrombospondin 2 (TSP2), an extracellular glycoprotein, have been identified using a PCR procedure. Species that show exon skipping and/or retained introns were revealed. In particular, a species was characterized in which exon 3 has been spliced from the sequence. This transcript is otherwise intact from exon 1A to exon 22. Transcripts which retain intron 1A, intron 1B, or intron 2 were also identified. Tissue-specific differences were evident in the transcripts with retained introns. In addition, a species which both retained intron 1B and skipped exon 3 was revealed. The existence of these alternative transcripts of the human TSP2 gene could be important for understanding the diverse functions and tissue-specific expression implicated for TSP2.

Relative abundance of thrombospondin 2 and thrombospondin 3 mRNAs in human tissues.

The levels of thrombospondin 2 (TSP2) and thrombospondin 3 (TSP3) mRNAs in a variety of human tissues were determined by analysis of multiple-tissue mRNA dot blots. For TSP2 mRNA, aorta and fetal heart had the greatest relative abundance. High levels were also detected for muscle, fetal, endocrine, immune, and nerve tissues. The pattern of expression of TSP3 mRNA was very different: kidney, pituitary gland, trachea, uterus, and fetal kidney had the greatest abundance. In general, TSP3 mRNA was expressed at high levels in endocrine, muscle, and fetal tissues. In addition to the tissue-specific differences, a more even distribution of TSP3 mRNA among tissues was observed. The high relative abundance of the two mRNAs in a variety of tissues and the tissue-specific differences in expression could be significant for understanding the diverse roles implicated for TSP2 and TSP3.

Analysis of the promoter and transcription start sites of the human thrombospondin 2 gene (THBS2).

To identify features of the human thrombospondin 2 gene (THBS2) important for regulation of expression, the sequences of 5 kb of the promoter/5' flank and 3 kb of transcribed and intronic DNA were determined. Two repetitive sequences were found: an MLT1c element located 2.2 kb 5' of exon 1 and, further 5', 1.8 kb of a Tigger1 element. Putative transcription factor binding sites that might be significant for THBS2 regulation included p53, NF-kappaB, Spl, Myc-CF1, NF-Y, CF1, AP1, and GATA sites. Alignment of the promoter/5' flank sequence with the mouse Thbs2 promoter revealed 78% identity for a 450 bp region immediately upstream from the mouse transcription start site. No significant homology was detected between the human thrombospondin 2 and thrombospondin 1 promoters. Comparison of the THBS2 genomic and cDNA sequences revealed that, in contrast to Thbs2, exon 1 is divided into exons 1A and 1B by a small (93 bp) intron. The transcription start site was investigated by a PCR procedure and by 5' RACE, and yielded a size for exon 1A of at least 186 bp. Tissue-specific differences in transcription start sites were found, with transcript lengths in the order: fetal lung > adult lung > fetal brain. These results suggest that tissue-specific differences in expression of the THBS2 gene may be determined, in part, by selection of the transcription start site and resulting differences in the 5' untranslated region.

Structure and organization of the human metaxin gene (MTX) and pseudogene.

Metaxin encodes a mitochondrial protein and is an essential nuclear gene in mice. The cDNA sequence and genomic organization of the human metaxin gene (MTX) have now been determined. MTX is 6 kb and consists of eight protein-encoding exons. The gene is contiguous to thrombospondin 3 (THBS3) and to the pseudogene for glucocerebrosidase (psGBA), but it transcribed in a direction opposite to the latter two genes. Thus, MTX and THBS3 share a common promoter region and are transcribed convergently, whereas MTX and psGBA are transcribed convergently and have closed apposed polyadenylation sites. Human metaxin contains 317 amino acids and is 91.5% identical to mouse metaxin. Metaxin is rich in leucine (14.2%) and in basic (12.9%) and acidic (12.0%) amino acids. The predicted protein lacks an amino-terminal signal sequence and N-glycosylation sites, but contains a putative transmembrane domain near its carboxy terminus. A DNA duplication has led to a direct repeat and the evolution of a pseudogene for GBA. A pseudogene for metaxin (psMTX) is also located within the 16 kb of DNA separating GBA from psGBA. The psMTX sequence is nearly identical to the 3' part of exon 2 through exon 8 of MTX, and both the intronic and the 3'-flanking sequences are highly conserved. Thus, there is a 278 amino acid open reading frame that is 97.8% identical to metaxin. However, psMTX lacks the first intron and promoter present in MTX, and at least in liver, the pseudogene is not expressed.

Structure and organization of the human thrombospondin 3 gene (THBS3).

The promoter/5' flank sequence, cDNA sequence, and exon/intron structures of the human thrombospondin 3 (THBS3) gene have been determined. THBS3 cDNA clones were obtained by PCR amplification of human fetal lung cDNA using THBS3-specific primers. Analysis of cDNA and genomic sequences showed the THBS3 gene to be composed of 23 exons, 1 more than the number of exons in the previously characterized mouse TSP3 gene. The additional exon results from the division of mouse exon F into exons F1 and F2. The cDNA encodes a polypeptide of 956 amino acids that is highly acidic, with a clustering of acidic side chains in the third quarter of the polypeptide. This region corresponds to seven type III (Ca(2+)-binding) repeats, a feature shared with other thrombospondins. In addition to these type III repeats, four type II (EGF-like) repeats and NH2- and COOH-terminal domains are present in thrombospondin 3. The THBS3 and mouse TSP3 genes differ in intron sizes, but exon sequences and sizes and positions of insertion of introns are conserved to a high degree. The structural organization of the THBS3 gene is of interest because of its close proximity to that of metaxin, with which it shares a common promoter sequence, and to the gene encoding glucocerebrosidase, a deficiency in which causes Gaucher disease.

ADPribosylation of nuclear proteins labeled with [3H]adenosine: changes during the HeLa cycle.

Cell cycle variations in the modification of histones and nonhistones by ADPribosylation were investigated. Proteins of HeLa interphase nuclei and metaphase chromosomes were radioactively labeled in vivo with [3H]adenosine. Histones of metaphase chromosomes were extensively modified by ADPribosylation, with H2B, H2A and H4 being predominant acceptors of [3H]adenosine label. For histones of interphase nuclei from synchronized cells, the highest level of 3H labeling was observed by two-dimensional gel electrophoresis to occur in S phase. The minimum level was noted in G1 phase. ADPribosylation of histones is, however, significant during all phases of the cell cycle. These conclusions were confirmed by experiments using [32P]NAD. The results with the specific inhibitor of ADPribosylation, 3-aminobenzamide, and with snake venom phosphodiesterase indicated that the radioactive isotopes were incorporated as ADPribose. Two-dimensional gels of HeLa nonhistones labeled with [3H]adenosine showed strikingly different patterns for interphase and metaphase samples. Over 100 ADPribosylated species were found for interphase nuclei, but poly(ADPribose) polymerase was the only major acceptor for metaphase chromosomes. A simple pattern was also revealed for nuclear scaffolds, with the 'lamins' and poly(ADPribose) polymerase being identifiable as modified species.

Factors influencing ADP-ribosylation differences between chromosomal proteins of interphase and metaphase HeLa cells.

Fundamental differences were previously discovered in the ADP-ribosylation of proteins from metaphase chromosomes and interphase nuclei of HeLa cells. The number of modified nonhistone species was found to be dramatically reduced for metaphase chromosomes. An investigation has therefore been made of factors which could influence, and therefore be responsible for, this change in ADP-ribosylation during the cell cycle. Modified proteins were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing mitotic and interphase samples from permeabilized cells that had been incubated with [32P]NAD. Whole cells showed a difference between interphase and metaphase similar to that for isolated nuclei and chromosomes. Chromosome expansion, disruption of chromosomes or nuclei, DNA nicking, and cellular growth activity significantly changed the incorporation of 32P label. Inhibitors of protein, RNA, and DNA synthesis did not, however, greatly affect ADP-ribosylation. The pattern of labeled species was not altered by the presence of nonradioactive NAD, though the extent of labeling declined. The results were not artifactually due to the procedure used to arrest cells in mitosis. Similar results were found with Novikoff rat hepatoma cells, demonstrating that the difference between metaphase and interphase is not confined to HeLa cells.

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.

Cell cycle variations in ADP-ribosylation of HeLa nuclear proteins.

Changes in ADP-ribosylation of nuclear proteins during the HeLa cell cycle were determined. Portions of synchronized cultures were withdrawn at intervals and cells were permeabilized by resuspension in hypotonic buffer containing detergents. Nuclear proteins were radioactively labeled by incubating samples with [32P]NAD. Modified species were resolved using one-dimensional and two-dimensional polyacrylamide gel electrophoresis. Measurements of the incorporation of [32P]NAD by permeabilized cells showed that ADP-ribosylation is a significant modification throughout the cell cycle. A twofold increase was detected during S phase. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gels revealed that many nuclear nonhistones are modified, though the major acceptors of 32P were the histones and a 116,000-Da species (poly(ADP-ribose) polymerase). The same modified proteins were present through the cell cycle, but densitometry of autoradiograms demonstrated a general increase in the level of incorporation in S phase. Autoradiograms of two-dimensional gels of nuclear proteins labeled with [32P]NAD were consistent with these results. Although nonhistones of isolated metaphase chromosomes show a substantial reduction in ADP-ribosylation, histone modification is essentially unchanged in metaphase.

Degree of conservation of HeLa interphase nonhistone antigens in metaphase and with chromatin from non-human cells.

Immunological procedures were applied to determine the degree of conservation of the nonhistone proteins of HeLa interphase chromatin. Polyacrylamide gels were overlaid with antiserum to HeLa interphase chromatin, and 125I-Protein A was used to detect bound antibodies. With two-dimensional gels, more than 80 interphase species were found as components of metaphase chromosomes. The degree of conservation of HeLa nonhistone antigens with other chromatin sources was calculated through densitometry of autoradiograms and stained gels. Chromatin was obtained from chicken erythrocytes, Novikoff rat hepatoma cells, and mouse L cells, and the presence of one-quarter to one-third of the nonhistone antigens of HeLa chromatin was demonstrated.

ADP-ribosylation of metaphase and interphase nonhistones using [3H]adenosine as a radioactive label.

ADP-ribosylation of HeLa nonhistone proteins was investigated by using [3H]adenosine as an in vivo radioactive label. The aim was to determine basic differences in the patterns of modification of interphase and metaphase nonhistones. Fluorography revealed a relatively small number of modified proteins for isolated metaphase chromosomes. In addition to the core histones, a protein of 116 kDa, which is identified as poly-(ADP-ribose) polymerase, was a primary acceptor of [3H]adenosine. Two-dimensional gels revealed a profound difference in the modification of metaphase and interphase nonhistones. For interphase nuclei, 3H label was distributed among a large number of nonhistone acceptors.

Decrease in ADP-ribosylation of HeLa non-histone proteins from interphase to metaphase.

Variations for non-histones in the ADP-ribosylating activities of interphase and metaphase cells were investigated. 32P-Labeled nicotinamide adenine dinucleotide ([32P]NAD), the specific precursor for the modification, was used to radioactively label proteins. Permeabilized interphase and mitotic cells, as well as isolated nuclei and chromosomes, were incubated with the label. One-dimensional and two-dimensional gels of the proteins of total nuclei and chromatin labeled with [32P]NAD showed more than 100 modified species. Changing the labeling conditions resulted in generally similar patterns of modified proteins, though the overall levels of incorporation and the distributions of label among species were significantly affected. A less complex pattern was found for nuclear scaffolds. The major ADP-ribosylated proteins included the lamins and poly(ADP-ribose) polymerase. Inhibitors of ADP-ribosylation were effective in preventing the incorporation of label by most non-histones. Snake venom phosphodiesterase readily removed protein-bound 32P radioactivity. A fundamentally different distribution of label from that of interphase nuclei and chromatin was found for metaphase chromosome non-histones. Instead of 100 or more species, the only major acceptor of label was poly(ADP-ribose) polymerase. This profound change during mitosis may indicate a structural role for ADP-ribosylation of non-histone proteins.

Relationship of the surface structure of metaphase chromosomes to the higher order organization of chromatin fibers.

Scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM), has been utilized to determine how the surface structure of mitotic chromosomes is related to the organization of the 30 nm chromosomal fibers. SEM revealed the surfaces of isolated, HeLa cell chromosomes to possess a knobby substructure with chromosomes prepared for EM in buffers containing 0.5-1.5 mM Mg2+. These projections had substantially greater widths (65-70 nm) than the underlying chromatin fibers. Reducing the Mg ion concentration to 0.05-0.15 mM resulted in the further expansion of the chromosomes, which flattened the chromosomes for SEM so the fibers became the dominant feature of the micrographs. The surface protuberances are interpreted as representing the peripheral tips of radial chromatin loops. The same procedure of slightly expanding chromosomes by decreasing the Mg2+ concentration in resuspension buffer was also utilized in a TEM, serial sectioning study. Longitudinal sections close to the central chromatid axis showed radially oriented fibers within the planes of the sections. This was replaced by a dot pattern when the longitudinal sections grazed the periphery of the chromatid. Transverse sections displayed more clearly the radial orientation of the fibers. A consistent picture emerges from applying SEM and TEM that supports the "radial loop" model for the primary mode of organization of chromatin fibers in metaphase chromosomes.

Variations in ADP-ribosylation of nuclear scaffold proteins during the HeLa cell cycle.

Cell cycle variations in ADP-ribosylation of nuclear scaffold proteins were determined. Nuclei of synchronized cells were isolated and labeled with [32P]NAD before nuclear scaffolds were obtained by digestion of DNA with DNase I and extraction of proteins with 2M NaCl. Autoradiograms revealed the three groups of "lamins" and a species identified as poly (ADP-ribose) polymerase to be the primary ADP-ribosylated proteins. The patterns of modification of nuclear scaffold proteins displayed similar features through the cell cycle. Radioactivity in the lamins increased from 20% in early-S phase to 40% in G1 phase of the next cell cycle.

Conservation of interphase chromatin nonhistone antigens as components of metaphase chromosomes.

The degree of conservation of HeLa interphase chromatin nonhistone antigens among the nonhistones of isolated metaphase chromosomes was determined with immunological procedures. Proteins were separated on SDS-polyacrylamide gels and electrophoretically transferred to diazophenylthioether (DPT)-paper, which was then overlaid with antiserum to chromatin from interphase nuclei. The bound antibodies were detected with 125I-labeled protein A. Alternatively, polyacrylamide gels were directly overlaid with antiserum and with 125I-protein A. Densitometry of autoradiograms and stained gels revealed the degree of conservation of nonhistone antigenic determinants from interphase to metaphase to be over 90% for chromatin.

ADP-ribosylation of nonhistone proteins during the HeLa cell cycle.

ADP-ribosylation of nonhistone proteins during the HeLa cell cycle was investigated. Proteins were radiolabeled by incubating interphase nuclei and mitotic cells with the specific precursor, [32P]NAD. Autoradiograms of two-dimensional gels of total nuclear nonhistone proteins showed a large number of modified species (more than 140). A complex pattern was also found for interphase chromatin. Nuclear scaffolds showed a simpler pattern of four major groups of modified species, which appeared to be the lamins and poly(ADP-ribose) polymerase. The labeling pattern for nonhistones of metaphase chromosomes was fundamentally different than with interphase nuclei. Autoradiograms were dominated by the incorporation of label into poly(ADP-ribose) polymerase.

Surface structure of isolated metaphase chromosomes.

The relationship between the surface protuberances of mitotic chromosomes isolated from HeLa cells and the underlying fiber organization was investigated by scanning electron microscopy (SEM). Chromosomes were isolated in the presence of 5.0 mM Mg2+ by a method which avoids the use of organic solvents and extremes of pH. Chromosomes in 5.0 mM Mg2+ are highly condensed with a relatively smooth surface structure. In 1.5 mM Mg2+, a knobby surface substructure became apparent, with the protuberances having a mean diameter of 691 +/- 96 A. The diameter was 647 +/- 76 A at a magnesium concentration of 0.5 mM, but was only 349 +/- 52 A at a concentration of 0.15 mM. In 0.05 mM Mg2+, the mean diameter had decreased to 299 +/- 47 A and the chromosomes had expanded such that the underlying fibers had become a predominant feature of the micrographs. The knobby appearance of the chromosomes most likely reflects a radial arrangement of the fibers, which loop back at the peripheries of the chromosomes.