Swapping the domains of exoribonucleases RNase II and RNase R: conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.     

RNA干扰技术抑制内源性绿色荧光蛋白的表达

目的建立稳定表达绿色荧光蛋白(GFP)的细胞株;构建短发夹RNA(shRNA)表达质粒并观察其对内源性GFP的抑制作用。方法转染pEGFP-N1至HepG2细胞,利用G418筛选获得稳定表达GFP的细胞株(HepG2.GFP);设计合成针对GFP基因的siRNA对应的DNA片段,插入转录载体pTZU6 1,构建shRNA表达载体pSHGFP,转染HepG2.GFP,荧光显微镜观察细胞荧光强度,以western blot检测GFP蛋白水平,以RT-PCR检测mRNA水平。结果利用PCR方法从HepG2.GFP细胞基因组DNA中检测到GFP基因;pSHGFP能够显著抑制该细胞中GFP的表达。结论GFP基因成功整合至HepG2细胞基因组中,pSHGFP能够显著抑制内源性GFP的表达,该系统能够用于RNA干扰机制等研究中。     

RBPMS不同剪接体的真核表达和亚细胞定位

目的：构建具有多种剪接形式的RNA结合蛋白（RBPMS）基因的真核表达载体,并在真核细胞中表达,确定不同形式的RBPMS在细胞中的定位。方法：采用PCR技术从人卵巢cDNA文库中扩增RBPMS基因的几种完整编码序列（命名为RBPl～RBP4）,克隆到带绿色荧光蛋白标签的pEGFP-C1表达载体上,转染人胚肾细胞293T,Western印迹鉴定RBPMS的表达,并利用激光共聚焦显微镜观察RBPMS不同剪接体在细胞中的定位。结果：限制性内切酶分析和DNA序列测定表明构建的重组表达载体正确,Western印迹实验证明RBP1～RBP4表达成功。通过激光共聚焦显微镜观察,RBP1／4围绕胞核在核膜的周围呈聚集状分布;RBP2则在细胞质和细胞核中均有分布,但会出现斑点状聚集;RBP3呈半月状紧密分布在细胞核周围;RBPMS中的RNA识别基序缺失后,这种现象消失,与空载体对照类似,在细胞核和细胞质中均有分布。结论：构建并表达了RBPMS基因的真核表达载体,RBPMS不同剪接体及RNA识别基序缺失后具有不同的亚细胞分布模式,提示具有不同的功能。     

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway.     

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.     

cDNA cloning and characterization of Drb1, a new member of RRM-type neural RNA-binding protein

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.     

Two ZBP1 KH domains facilitate beta-actin mRNA localization,granule formation,and cytoskeletal attachment

Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.     

Global signatures of protein binding on structured RNAs in Saccharomyces cerevisiae

Protein binding is essential to the transport,decay and regulation of almost all RNA molecules.However,the structural preference of protein binding on RNAs and their cellular functions and dynamics upon changing environmental conditions are poorly understood.Here,we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins(RBPs)and structured RNAs in yeast at single-nucleotide resolution.We found that on average,in terms of percent total lengths,~15%of mRNA untranslated regions(UTRs),~37%of canonical non-coding RNAs(ncRNAs)and~11%of long ncRNAs(lncRNAs)are bound by proteins.The RBP binding sites,in general,tend to occur at single-stranded loops,with evolutionarily conserved signatures,and often facilitate a specific RNA structure conformation in vivo.We found that four nucleotide modifications of tRNA are significantly associated with RBP binding.We also identified various structural motifs bound by RBPs in the UTRs of mRNAs,associated with localization,degradation and stress responses.Moreover,we identified>200 novel lncRNAs bound by RBPs,and about half of them contain conserved secondary structures.We present the first ensemble pattern of RBP binding sites in the structured non-coding regions of a eukaryotic genome,emphasizing their structural context and cellular functions.     

Lacustrine sedimentation in the diapir-controlled Miocene Bicorb Basin, eastern Spain

The Miocene Bicorb Basin is a small elongated basin developed by normal faulting of a thick Jurassic–Cretaceous carbonate succession and subsequent diapirism of Upper Triassic mudstones and evaporites. The basin fill comprises a sequence over 650 m thick formed by two units. The lower, alluvial unit consists of a complex alternation of conglomerates, sandstones, mudstones, and minor lacustrine limestones. The upper unit comprises marginal alluvial and lacustrine deposits in which seven lacustrine facies associations have been distinguished. The inner lacustrine deposits comprise mudstones and carbonates with minor evaporitic deposits. At the northeast and southwest basin edges, alluvial inputs led to the development of delta and fan-delta environments where sandstone and conglomerate deposits dominate. The latter stages of the lake evolution are represented by an expansive thick limestone sequence which close to the NW fault-bounded margin pass laterally to breccias. The hierarchical arrangement of the upper unit shows five different orders of sequences. The first-order sequence defines a major vertical trend of lake expansion whereas three second-order sequences are linked to major flooding-expansion episodes. All these sequences are related to the tectonic evolution of the diapir. Metre-scale, third- and fourth-order sequences and fifth-order sequences, which consist of finely laminated rhythmite deposits probably record climatically forced processes. The diapir–graben system evolution and the climatic variations have exerted major controls on the sequential arrangement and evolution of the lacustrine system. Both factors have also strongly influenced the changes in the gastropod assemblages.     

The syn-volcanic Naajaat lake, Paleocene of West Greenland

The Naajaat lake in the Nuussuaq Basin on Disko and Nuussuaq formed in a geological setting between cratonic crystalline Precambrian rocks overlain by Cretaceous sediments and an actively forming Paleocene volcanic province. The lacustrine deposits, shales as well as hyaloclastite breccias, accumulated in low-lying areas inundated by fresh water and sealed off from marine transgressions by a broad subaerial volcanic terrain. Foreset-bedded hyaloclastite breccias demonstrate water depths of up to 450 m, and the area of the lake was 2500 km² at its maximum extent. The lake probably existed for less than 0.5 million years. The lake received clay and silt from two provenance areas. Quartz contents of more than 25% in the majority of the sediment samples indicate that large amounts of material were continuously supplied to the lake from the crystalline terrain, whereas the volcanic terrain supplied smectite and mixed-layer minerals to the lake. High kaolinite contents stem from the crystalline or both provenance areas. The shales are characterized by high TOC (up to 11%), lack of pyrite, presence of terrestrial spores and pollen and lack of marine dinoflagellates. The lacustrine sediments rest on an erosional unconformity and its correlative conformity. The unconformity developed during the latest Cretaceous and Early Paleocene. Five stages are recognized in the geological development of the lake. Stages 1–4 are characterized by accumulation of hyaloclastite breccias, rise in lake level, and eventual transgression of subaerial terrains. The rises in lake level were caused by stemming of fluvial run-off behind the aggrading volcanic pile. Stage 5 corresponds to cessation of volcanic activity, a stable lake level, and progradation of clastic sediments, resulting in infilling of the lake. During the lacustrine transgression only sediment in suspension was transported into the central parts of the lake.     

Evidence for Late Glacial and Holocene environmental changes from subfossil assemblages in sediments of Lake Neuchâtel, Switzerland

Ostracod and diatom assemblages, mollusc remains, plus pollen and sediment stratigraphy from three cores taken at sites 40, 80 and 135 m deep in Lake Neuchâtel, Switzerland, show distinct changes since deglaciation. Shifts are interpreted in terms both of climatic change and major changes in the catchment input caused by the Aar River entering or bypassing Lake Neuchâtel. Three palaeoenvironmental phases are recognized: (1) the Late Glacial, with the inflow of the river before 10,000 yr B.P.; (2) the early to mid-Holocene, with temporary bypassing of the river between 10,000 and 4850 yr B.P.; and (3) the mid- to late Holocene, without river input after approx. 4850 yr B.P. During periods with river input, rhythmites were deposited. Ostracods indicate a well oxygenated hypolimnion, suggesting deep ventilation enhanced by the river inflow. The diatom assemblage indicates eutrophic to mesotrophic conditions and is dominated by generally well preserved periphytic species. The high number of unidentifiable pollen suggests oxidation during transport in the river. During periods without river input, a massive calcareous silt with abundant authigenic calcite was deposited, diatoms indicate a change to mesotrophic and oligotrophic conditions, and decreased sedimentation rates and higher alkalinity enhanced the dissolution of diatoms. Terrestrial and littoral molluscs as well as periphytic diatoms indicate erosion of the shore when the lake level rose or fell with onset or offset of the river inflow. Before 12,600 yr B.P., the patchy vegetation cover led to intensive soil erosion and high sedimentation rates. The lake was still frozen during the winter months. After approx. 12,600 yr B.P. the sediment changed from clastic rhythmites to thinly bedded, non-glacial rhythmites, the vegetation cover became denser, the abundance of ostracods increased, and molluscs started to colonize the profundal zone of the lake. The Younger Dryas (approx. 10,700–10,000 yr B.P.) displays maximum abundance of ostracods, indicating deep ventilation. The transition to the Holocene is characterized by a change in the ostracod assemblage and the river bypassed the lake between approximately 10,000 and 8900 yr B.P. When the river reentered the lake after approximately 8900 yr B.P., a new ostracod species appeared. After two short periods of river bypassing between approximately 7200 and 6950 yr B.P., diatoms indicate a higher trophic level about 6000–5000 yr B.P. associated with higher erosion rates and increased nutrient input due to Neolithic agricultural activities. After approximately 4850 yr B.P. the river bypassed the lake again and the trophic level decreased. In contrast to the early to mid-Holocene periods of decreased oxygen supply, the mid- to late Holocene is characterized by deep ventilation. This is attributed to the northward retreat of the polarfront and enhanced westerlies consequent to decreased insolation after 6000 yr B.P. The change of the river flow pattern correlates with glacier oscillations and may suggest a climatic control. The final cessation of the inflow is attributed to diversion of the river consequent to isostatic uplift and late Neolithic clearance activity.     

Vibracoring: a new method for coring deep lakes

A major limitation facing lacustrine researchers is an inability to take long (6 to 18 m), continuous sediment cores in water depths greater than several metres at a low cost. These problems have limited the advancement of palaeolimnology and Quaternary stratigraphy. Working from a frozen lake surface, new developments make it possible to take 18 m long vibracores in water depths of at least 55 m. High-frequency low-amplitude vibrations are transferred down a series of 6 m long, 7.6 cm diameter core tubes (3 inch aluminum irrigation pipe) rigidly coupled end-to-end. Vibrations liquify sediment adjacent to the core tube walls, thus allowing penetration of the sediment. A core catcher prevents sediment from extruding out of the tube end during extraction. This cost-effective and labour efficient (two persons) coring system has been achieved by developing rigid core tube couplers, a hand-powered drum cable hoist and a set of operation procedures. The hoist cable end is attached to a clamp 1 m above the core catcher and at each coupler along the pipe string at 6 m intervals. Lifting or lowering using a drum cable hoist allows for quick coupling and decoupling of core tubes. Although vibracoring is an improvement over other systems, it still has portability limitations because of the bulk and weight of the equipment and large amount of pipe required for deep coring. Three sleds are required to transport equipment from the nearest vehicle access to the coring sites.     

Severe drought in the early Holocene (10,000–6800 BP) interpreted from lake sediment cores, southwestern Alberta, Canada

Six lake sediment vibracores from the foothills and mountain valleys of southwestern Alberta were analysed on the basis of AMS radiocarbon dates, tephrochronology and sediment geochemistry. From these results, the magnitude, timing and duration of the early Holocene warm period are presented. At about 10,000 BP, immediately following the Younger Dryas cold period, climate warmed dramatically, precipitation decreased and surface evaporation increased. Previous research has identified this warm interval, but new results have improved resolution of regional scale effects, specific timing and severity. Sedimentation changed from extra-basinal clastic to intra-basinal organic between 10,000 and 9400 BP. Changes in subalpine lakes from sand/silt deposition to biogenic carbonate precipitation suggest decreases in suspended sediment load caused by complete ablation of glacial sediment sources. Peat which formed in lakes of less than 4 m (present depth) indicates climate-induced lake level lowering in the foothills. Water depth and stratigraphic position of the peat suggest that regional water table levels decreased by up to 6.5 m. At Cartwright Lake, an erosional unconformity 6.5 m below the modern lake surface indicates the lake had completely dried out either during or immediately after Mazama tephra time (6800 BP).     

Surface analytical techniques applied to calcite: evidence of solid-state diffusion and implications for isotope methods

Surface analytical techniques have become common tools for physicists and materials scientists for making direct observations at the molecular scale but they can also provide Earth Scientists with new information about mineral/fluid interfaces. High resolution surface analysis compliments our traditional methods for analysing the bulk of solids and solutions which can give us, for example, a more complete picture of the geochemical processes that affect the mineral grains in sediments. Stable isotope methods have become widely used for dating and palaeoclimate studies but they rely on the assumption of a closed system. Artifacts resulting from carbonate mineral recrystallization can be avoided by careful sampling but ion by ion replacement can significantly alter the composition of a solid without leaving visible traces. Calcite has long been known to take up substituting ions into its atomic structure. Recent evidence gained from surface analysis shows Cd²⁺ and Zn²⁺, that had been adsorbed or precipitated on calcite surfaces, moves into calcite at the rate of nanometers per month, apparently by solid-state diffusion. No fractures that could serve as conduits from surface to bulk have ever been observed at the micrometer to nanometer scale on the single crystals used for these experiments. Mixing within the top few surface layers by reprecipitation during exposure to the humidity in air can account for some incorporation, but the evidence collected so far does not explain the exchange of position for ions beyond about ten calcite monolayers. With similar rates of movement, other trace components, such as K⁺, Na⁺, Cl^- and F^-, have been observed to move out of bulk calcite and to accumulate in discrete crystallites on surfaces exposed to air. Such mobility may be particular to these near-perfect, Iceland spar crystals and the ions investigated, but if O and C also move into and out of bulk calcite at similar rates, the integrity of isotope ratios from carbonate minerals, even from non-diagenetic environments, may be questionable.     

Palaeolimnological investigations in Poland based on Cladocera (Crustacea)

The results of subfossil Cladocera analysis obtained from deposits of 26 lakes throughout Poland have been presented. For years throughout Poland a broad range of palaeolimnological studies have been conducted. Their aim is to discover the historical development of water basins and climatic changes in the course of the last 13,000 years and also to investigate the gradual influence of man on the ecosystem with particular emphasis on the last few decades. The hitherto obtained results of palaeolimnological studies, including the results of Cladocera analysis, have yielded information about the development of lakes and provided the basis for plans of certain reservoirs and their basins. Over the past years, analysis of subfossil Cladocera has become one of the leading palaeolimnological methods. The results of detailed qualitative and quantitative analysis of Cladocera remains from sediments of 15 lakes allowed for a reconstruction of the lakes' development. The littoral and limnetic regions, changing trophy, from oligotrophy to advanced eutrophication, changes in pH and fluctuation of water levels, as well as changes resulting from settlement — from Mesolithic and Neolithic through Medieval times until the present — have been reconstructed. For individual lakes, phases of Cladocera development and periods of eutrophication were indicated which partially correlated with certain pollen zones as well as periods of human impact and geochemical effects.     

The laminated sediments of Loch Ness, Scotland: Preliminary report on the construction of a chronology of sedimentation and its potential use in assessing Holocene climatic variability

Two sediment cores, ca. 6 m long, have been recovered from the north basin of Loch Ness, Scotland. Each consists of ca. 4.5 m of laminated gyttja, terminating in a basal grey, unlaminated clay. A suite of three ¹⁴C AMS dates have been obtained, and place the base of the gyttja at ca. 9500 yr B.P. Investigations into the structure, composition and formation of the laminae are in progress. Analysis by Backscatter Scanning Electron Microscopy (BSEM) has demonstrated that they consist of couplets comprising dark, clay-rich sediments thought to be deposited from spring to autumn, and pale, silt-rich layers believed to represent sedimentation through winter. The laminae are thus thought to record incidence and intensity of streamflow into the Loch over the past nine millennia, and thus illustrate environmental change over the region for most of the Holocene. X-ray densitometry has been utilised in order to count the laminations and test the hypothesis that they are varves. It has, however, proved difficult to obtain a continuous sequence of countable laminations, although it has been possible to construct a fragmented, floating chronology which indicates that the hypothesis may be correct. Examination of lamination thickness reveals that although the average rate of sedimentation throughout the time periods studied seems to have remained fairly constant, significant variations have occurred. As Loch Ness is located on the northwest oceanic fringe of Europe, any climatic signal which the laminations contain will be closely related to even larger scale events over and within the North Atlantic Ocean, which is a major controller of global climate. Preliminary statistical investigation of sequences of laminae is being carried out in order to search for periodicity of sedimentation which may then be related to appropriate climatic indices.     

The use of the microalga Botryococcus in the interpretation of lacustrine environments at the Jurassic–Cretaceous transition in Sweden

Ultrastructural studies of developmental stages, shapes and size of fossil microalgal Botryococcus colonies, found in the Vitabäck clays at Eriksdal in southern Sweden, have been carried out. The results of this study, by analogy with the modern alga, suggest that for these lacustrine environments short limited periods of stable, uniform climatic conditions were interspersed with longer periods of seasonal climatic changes during the deposition of the Jurassic–Cretaceous transition sediments in Sweden.     

Late glacial and early Holocene palaeoenvironmental changes in Geneva Bay (Lake Geneva, Switzerland)

Detailed interdisciplinary investigations demonstrate that Geneva Bay (Lake Geneva) sediments clearly record important palaeoenvironmental and palaeoclimatic changes occurred during the Late glacial and early Holocene. Sediments are in fact differentiated by changes in texture, mineralogical and geochemical composition. Distal turbidite and glacial rhythmite deposition associated with wind-transported sediment supply dominate during the Oldest Dryas. These were replaced during the Allerød by detrital settling of sediment from turbid water and by endogenic calcite precipitation. The Younger Dryas climate reversal (for the first time in Lake Geneva well documented by a pollen record) was characterized by an increase in detrital supply owing to increased run-off from the bay slopes surrounding and within the catchment area, caused by thinning of vegetation cover. A brief pause in endogenic precipitation related to decreased productivity also occurs at this time. Endogenic carbonate sedimentation abruptly resumed at the start of the Preboreal biozone in response to the rapid global climatic warming. In the middle Preboreal, renewed detrital sedimentation is interpreted as the sedimentological response to increased erosion in high-altitude regions of the catchment area, caused by a minor early Holocene cooling phase (Preboreal oscillation), and interrupts the trend towards increasing endogenic calcite precipitation. Favourable limnic conditions are reached during the late Preboreal, when diatom–calcite rhythmites begin to form. Stable isotope analysis (δ¹⁸O, δ¹³C) in bulk carbonate highlight the transition from clastic-dominated to endogenic-dominated sedimentation. Endogenic calcite deposition continues during the Boreal biozone, occasionally interrupted by local high-energy sedimentary processes (wave-induced erosion and reworking of littoral deposits) enhanced by progressive lake-level lowering. During the Older Atlantic biozone, sedimentation was mainly dominated by low-energy deposition, creating enhanced conditions for the development of benthic fauna. We will discuss the factors that make Geneva Bay an important site for recording the Late glacial–Holocene climate and environmental changes. The comparison with other Swiss Plateau lacustrine systems allowed us to emphasize the role played by the particular geographical and morphological setting of the site investigated and links with climate-sensitive regions in the catchment area.