Cloning and characterization of the full length cDNA and promoter of mouse tissue transglutaminase

Transglutaminases are a family of enzymes that catalyze the covalent cross-linking of proteins through the formation of $\varepsilon$-($\gamma$-glutaminyl)-lysyl isopeptide bonds. Tissue transglutaminase (Tgase) is an intracellular enzyme which is expressed in terminally differentiated and senescent cells and also in cells undergoing apoptotic cell death. To characterize this enzyme and examine its relationship with other members of the transglutaminase family, cDNAs, the first two exons of the gene and 2 kb of the 5$\sp\prime$ flanking region, including the promoter, were isolated. The full length Tgase transcript consists of 66 bp of 5$\sp\prime$-UTR (untranslated) sequence, an open reading frame which encodes 686 amino acids and 1400 bp of 3$\sp\prime$-UTR sequence. Alignment of the deduced Tgase protein sequence with that of other transglutaminases revealed regions of strong homology, particularly in the active site region.^ The Tgase cDNA was used to isolate and characterize a genomic clone encompassing the 5$\sp\prime$ end of the mouse Tgase gene. The transcription start site was defined using genomic and cDNA clones coupled with S1 protection analysis and anchored PCR. This clone includes 2.3 kb upstream of the transcription start site and two exons that contain the first 256 nucleotides of the mouse Tgase cDNA sequence. The exon intron boundaries have been mapped and compared with the exon intron boundaries of three members of the transglutaminase family: human factor XIIIa, the human keratinocyte transglutaminase and human erythrocyte band 4.1. Tissue Tgase exon II is similar to comparable exons of these genes. However, exon I bears no resemblance with any of the other transglutaminase amino terminus exons.^ Previous work in our laboratory has shown that the transcription of the Tgase gene is directly controlled by retinoic acid and retinoic acid receptors. To identify the region of the Tgase gene responsible for regulating its expression, fragments of the Tgase promoter and 5$\sp\prime$-flanking region were cloned into the chloramphenicol actetyl transferase (CAT) reporter constructs. Transient transfection experiments with these constructs demonstrated that the upstream region of Tgase is a functional promoter which contains a retinoid response element within a 1573 nucleotide region spanning nucleotides $-$252 to $-$1825. ^

Interactions and structural analysis of the zinc -binding motif in decorin, a small leucine rich proteoglycan in extracellular matrix

Decorin, a dermatan/chondroitin sulfate proteoglycan, is ubiquitously distributed in the extracellular matrix (ECM) of mammals. Decorin belongs to the small leucine rich proteoglycan (SLRP) family, a proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. The decorin core protein appears to mediate the binding of decorin to ECM molecules, such as collagens and fibronectin. It is believed that the interactions of decorin with these ECM molecules contribute to the regulation of ECM assembly, cell adhesions, and cell proliferation. These basic biological processes play critical roles during embryonic development and wound healing and are altered in pathological conditions such as fibrosis and tumorgenesis. ^ In this dissertation, we discover that decorin core protein can bind to Zn2+ ions with high affinity. Zinc is an essential trace element in mammals. Zn2+ ions play a catalytic role in the activation of many enzymes and a structural role in the stabilization of protein conformation. By examining purified recombinant decorin and its core protein fragments for Zn2+ binding activity using Zn2+-chelating column chromatography and Zn2+-equilibrium dialysis approaches, we have located the Zn2+ binding domain to the N-terminal sequence of the decorin core protein. The decorin N-terminal domain appears to contain two Zn2+ binding sites with similar high binding affinity. The sequence of the decorin N-terminal domain does not resemble any other reported zinc-binding motifs and, therefore, represents a novel Zn 2+ binding motif. By investigating the influence of Zn2+ ions on decorin binding interactions, we found a novel Zn2+ dependent interaction with fibrinogen, the major plasma protein in blood clots. Furthermore, a recombinant peptide (MD4) consisting of a 41 amino acid sequence of mouse decorin N-terminal domain can prolong thrombin induced fibrinogen/fibrin clot formation. This suggests that in the presence of Zn2+ the decorin N-terminal domain has an anticoagulation activity. The changed Zn2+-binding activities of the truncated MD4 peptides and site-directed mutagenesis generated mutant peptides revealed that the functional MD4 peptide might contain both a structural zinc-binding site in the cysteine cluster region and a catalytic zinc site that could be created by the flanking sequences of the cysteine cluster region. A model of a loop-like structure for MD4 peptide is proposed. ^

Evolution of cis-regulatory elements in a repetitive sequence adjacent to a sea urchin aboral ectoderm -specific gene

The creation, preservation, and degeneration of cis-regulatory elements controlling developmental gene expression are fundamental genome-level evolutionary processes about which little is known. In this study, critical differences in cis-regulatory elements controlling the expression of the sea urchin aboral ectoderm-specific spec genes were identified and explored. In genomes of species within the Strongylocentrotidae family, multiple copies of a repetitive sequence element termed RSR were present, but RSRs were not detected in genomes of species outside Strongylocentrotidae. RSRs are invariably associated with spec genes, and in Strongylocentrotus purpuratus, the spec2a RSR functioned as a transcriptional enhancer displaying greater activity than RSRs from the spec1 or spec2c paralogs. Single base-pair differences at two cis-regulatory elements within the spec2a RSR greatly increased the binding affinities of four transcription factors: SpCCAAT-binding factor at one element and SpOtx, SpGoosecoid, and SpGATA-E at another. The cis-regulatory elements to which SpCCAAT-binding factor, SpOtx, SpGoosecoid, and SpGATA-E bound were recent evolutionary acquisitions that could act either to activate or repress transcription, depending on the cell type. These elements were found in the spec2a RSR ortholog in Strongylocentrotus pallidus but not in the RSR orthologs of Strongylocentrotus droebachiensis or Hemicentrotus pulcherrimus. These results indicate that spec genes exhibit a dynamic pattern of cis-regulatory element evolution while stabilizing selection preserves their aboral ectoderm expression domain. ^

Expression regulation of AP -2 gamma gene

AP-2γ is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placenta development. In an effort to identify factors regulating AP-2γ gene expression we isolated and characterized the promoter and 5′ flanking region of the mouse and human AP-2γ genes. The transcription start site of the mouse AP-2γ gene was mapped by primer extension and 5′ RACE. Transient gene transfer studies showed that basal promoter activity resides within a highly conserved ∼200 by DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp and AP-2 binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2γ gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and non-trophoblast cells, demonstrating the functional importance of these sites in AP-2γ gene expression. ^ Mutational analysis of the 5′-flanking region revealed a 117-bp positive regulatory region of the mouse AP-2γ gene located between −5700 and −5583 upstream of the transcription start site. This 117-bp positive regulatory element provided approximately 7-fold enhancement of reporter gene expression in cultured trophoblast cells. A C/EBP-Sp1 transcription factor-binding module is located in this DNA sequence. Electrophoretic mobility shift assays demonstrated that transcription factors Sp1, Sp3 and C/EBP bind to the enhancer element. Mutation of each protein-binding site reduced the enhanced expression significantly. Mutagenesis assays showed that two other protein-binding sites also contribute to the enhancer activity. In summary, we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter region and contribute to basal promoter activity. We have identified a 117-bp positive regulatory element of AP-2γ gene, and we have shown that Sp and C/EBP proteins bind to the cis -regulatory elements and contribute to the enhanced gene expression. ^

Analyses of a Holocene soil/loess sequence in Southern Siberia

We conducted a high-resolution study of a unique Holocene sequence of wind-blown sediments and buried soils in Southern Siberia, far from marine environment influences. This was accomplished in order to assess the difference between North Atlantic marine and in-land climate variations. Relative wind strength was determined by grain size analyses of different stratigraphic units. Petromagnetic measurements were performed to provide a proxy for the relative extent of pedogenesis. An age model for the sections was built using the radiocarbon dating method. The windy periods are associated with the absence of soil formation and relatively low values of frequency dependence of magnetic susceptibility (FD), which appeared to be a valuable quantitative marker of pedogenic activity. These events correspond to colder intervals which registered reduced solar modulation and sun spot number. Events, where wind strength was lower, are characterized by soil formation with high FD values. Spectral analysis of our results demonstrates periodic changes of 1500, 1000 and 500 years of relatively warm and cold intervals during the Holocene of Siberia. We presume that the 1000 and 500 year climatic cycles are driven by increased solar insolation reaching the Earth surface and amplified by other still controversial mechanisms. The 1500 year cycle associated with the North Atlantic circulation appears only in the Late Holocene. Three time periods - 8400-9300 years BP, 3600-5100 years BP, and the last ~250 years BP - correspond to both the highest sun spot number and the most developed soil horizons in the studied sections

(Table 1) Chemical composition of volcanic rocks from the Mariisky sequence and extrusive vent rocks of the Mary Cape, Schmidt Peninsula (North Sakhalin)

Early Cretaceous volcanic rocks of the Mariisky sequence and Early Cenozoic extrusive-vent rocks of the Mary Cape are exposed at the most northwest of the Schmidt Peninsula, North Sakhalin. In chemical composition, all the rocks are subdivided into four groups. Three groups include volcanic rocks of the Mariisky sequence, which consists, from bottom to top, of calc-alkaline rocks, transitional calc-alkaline-tholeiite rocks, and incompatible element-depleted tholeiites. These rocks show subduction geochemical signatures and are considered as a fragment of the Moneron-Samarga island arc system. Trace-element modeling indicates their derivation through successive melting of a garnet-bearing mantle and garnet-free shallower mantle sources containing amphibole; pyroxene; and, possibly, spinel. The mixed subduction and intra-plate characteristics of the extrusive vent rocks of the Mary Cape attest to their formation in a transform continental margin setting.

Depths and ages of seismic sequence boundaries in ODP Leg 166 holes

High-resolution, multichannel seismic data collected across the Great Bahama Bank margin and the adjacent Straits of Florida indicate that the deposition of Neogene-Quaternary strata in this transect are controlled by two sedimentation mechanisms: (1) west-dipping layers of the platform margin, which are a product of sea-level-controlled, platform-derived downslope sedimentation; and (2) east- or north-dipping drift deposits in the basinal areas, which are deposited by ocean currents. These two sediment systems are active simultaneously and interfinger at the toe-of-slope. The prograding system consists of sigmoidal clinoforms that advanced the margin some 25 km into the Straits of Florida. The foresets of the clinoforms are approximately 600 m high with variable slope angles that steepen significantly in the Pleistocene section. The seismic facies of the prograding clinoforms on the slope is characterized by dominant, partly chaotic, cut-and-fill geometries caused by submarine canyons that are oriented downslope. In the basin axis, seismic geometries and facies document deposition from and by currents. Most impressive is an 800-m-thick drift deposit at the confluence of the Santaren Channel and the Straits of Florida. This "Santaren Drift" is slightly asymmetric, thinning to the north. The drift displays a highly coherent seismic facies characterized by a continuous succession of reflections, indicating very regular sedimentation. Leg 166 of the Ocean Drilling Program (ODP) drilled a transect of five deep holes between 2 and 30 km from the modern platform margin and retrieved the sediments from both the slope and basin systems. The Neogene slope sediments consist of peri-platform oozes intercalated with turbidites, whereas the basinal drift deposits consist of more homogeneous, fine-grained carbonates that were deposited without major hiatuses by the Florida Current starting at approximately 12.4 Ma. Sea-level fluctuations, which controlled the carbonate production on Great Bahama Bank by repeated exposure of the platform top, controlled lithologic alternations and hiatuses in sedimentation across the transect. Both sedimentary systems are contained in 17 seismic sequences that were identified in the Neogene-Quaternary section. Seismic sequence boundaries were identified based on geometric unconformities beneath the Great Bahama Bank. All the sequence boundaries could be traced across the entire transect into the Straits of Florida. Biostratigraphic age determinations of seismic reflections indicate that the seismic reflections of sequence boundaries have chronostratigraphic significance across both depositional environments.

X-ray fluorescence scanning of discrete samples on the Schwalbenberg II loess-paleosol sequence, raw data, magnetic susceptibility, organic carbon and U-ratio as grainsize index

The Schwalbenberg II loess-paleosol sequence (LPS) denotes a key site for Marine Isotope Stage (MIS 3) in Western Europe owing to eight succeeding cambisols, which primarily constitute the Ahrgau Subformation. Therefore, this LPS qualifies as a test candidate for the potential of temporal high-resolution geochemical data obtained X-ray fluorescence (XRF) scanning of discrete samplesproviding a fast and non-destructive tool for determining the element composition. The geochemical data is first contextualized to existing proxy data such as magnetic susceptibility (MS) and organic carbon (Corg) and then aggregated to element log ratios characteristic for weathering intensity [LOG (Ca/Sr), LOG (Rb/Sr), LOG (Ba/Sr), LOG (Rb/K)] and dust provenance [LOG (Ti/Zr), LOG (Ti/Al), LOG (Si/Al)]. Generally, an interpretation of rock magnetic particles is challenged in western Europe, where not only magnetic enhancement but also depletion plays a role. Our data indicates leaching and top-soil erosion induced MS depletion at the Schwalbenberg II LPS. Besides weathering, LOG (Ca/Sr) is susceptible for secondary calcification. Thus, also LOG (Rb/Sr) and LOG (Ba/Sr) are shown to be influenced by calcification dynamics. Consequently, LOG (Rb/K) seems to be the most suitable weathering index identifying the Sinzig Soils S1 and S2 as the most pronounced paleosols for this site. Sinzig Soil S3 is enclosed by gelic gleysols and in contrast to S1 and S2 only initially weathered pointing to colder climate conditions. Also the Remagen Soils are characterized by subtle to moderate positive excursions in the weathering indices. Comparing the Schwalbenberg II LPS with the nearby Eifel Lake Sediment Archive (ELSA) and other more distant German, Austrian and Czech LPS while discussing time and climate as limiting factors for pedogenesis, we suggest that the lithologically determined paleosols are in-situ soil formations. The provenance indices document a Zr-enrichment at the transition from the Ahrgau to the Hesbaye Subformation. This is explained by a conceptual model incorporating multiple sediment recycling and sorting effects in eolian and fluvial domains.

Secondary mineral assemblages in a volcanic sequence of ODP Hole 104-642E

Mineralogical and geochemical analyses of alteration products from upper and lower volcanic series recovered during ODP Leg 104 reveal variations both in composition and order of crystallization of clay minerals vesicles and voids filling and replacing glass. These results provide information about successive alteration stages of rocks and interlayered volcaniclastic sediments. The first stage, related to initial basalt-seawater interaction, is characterized by development of Fe-smectites, especially Fe-rich saponite. A second stage of intermittently superimposed subaerial weathering is marked by iron-oxides-halloysite-kaolinite formation. The third episode, interpreted as hydrothermal on the basis of O-isotopic data, is defined by postburial coprecipitation of Fe-poor, Mg-rich saponite and celadonite. A distinct final and pervasive hydrothermal stage, occurring mainly in the lower series and dominated by Al-smectites-zeolites assemblage, indicates changes toward a more reducing alteration environment.

Tab. 1: Meerfelder Maar Drilling B4, stratigraphy and sequence of the lake sediments (pdf 215 kB)

A core from Meerfelder Maar, with a basal age of 29,000 years, provides a continuous sedimentary sequence from Late-Glacial times to the present. It includes the stratigraphical marker of the Laach Pumice Tuff. Sedimentological, geochemical, palynological, palaeobiological, palaeomagnetic and palaeontological analyses permit reconstructions of the history of the lake and its catchment area, and hence of the climate of the region, to be made. The discovery of Middle Oligocene marine, detrital fossils in the maar sediments provides insights into the palaeogeography of the Eifel region during Tertiary times.