A systematic expression analysis implicates Plexin-B2 and its ligand Sema4C in the regulation of the vascular and endocrine system

Plexins serve as receptors for semaphorins and play important roles in the developing nervous system. Plexin-B2 controls decisive developmental programs in the neural tube and cerebellum. However, whether Plexin-B2 also regulates biological functions in adult nonneuronal tissues is unknown. Here we show by two methodologically independent approaches that Plexin-B2 is expressed in discrete cell types of several nonneuronal tissues in the adult mouse. In the vasculature, Plexin-B2 is selectively expressed in functionally specialized endothelial cells. In endocrine organs, Plexin-B2 localizes to the pancreatic islets of Langerhans and to both cortex and medulla of the adrenal gland. Plexin-B2 expression is also detected in certain types of immune and epithelial cells. In addition, we report on a systematic comparison of the expression patterns of Plexin-B2 and its ligand Sema4C, which show complementarity or overlap in some but not all tissues. Furthermore, we demonstrate that Plexin-B2 and its family member Plexin-B1 display largely nonredundant expression patterns. This work establishes Plexin-B2 and Sema4C as potential regulators of the vascular and endocrine system and provides an anatomical basis to understand the biological functions of this ligand-receptor pair.     

Green fluorescent proteins with short half-lives as reporters in Dictyostelium discoideum

 We describe two modifications of the popular reporter green fluorescent protein (GFP) which have short half-lives in our system, the cellular slime mould Dictyostelium discoideum. One of these bears an N-terminal ubiquitin; this GFP was originally planned to be a substrate of the ”N-end-rule” pathway, but deubiquitination does not seem to occur, and a degradation by the UFD (ubiquitin-fusion-degradation pathway seems more probable. The protein half-life is about 3–5 h. The second construct has an N-terminus derived from the L11 ribosomal protein; it is transported to the nucleus and broken down much more rapidly than the ubiquitin fusion (protein half-life about 30 min). We show examples of the use of these reporters in the study of gene expression in Dictyostelium. Received: 20 April 1998 / Accepted: 23 August 1998     

Support vector machines for separation of mixed plant-pathogen EST collections based on codon usage

MOTIVATION: Discovery of host and pathogen genes expressed at the plant-pathogen interface often requires the construction of mixed libraries that contain sequences from both genomes. Sequence identification requires high-throughput and reliable classification of genome origin. When using single-pass cDNA sequences difficulties arise from the short sequence length, the lack of sufficient taxonomically relevant sequence data in public databases and ambiguous sequence homology between plant and pathogen genes. RESULTS: A novel method is described, which is independent of the availability of homologous genes and relies on subtle differences in codon usage between plant and fungal genes. We used support vector machines (SVMs) to identify the probable origin of sequences. SVMs were compared to several other machine learning techniques and to a probabilistic algorithm (PF-IND) for expressed sequence tag (EST) classification also based on codon bias differences. Our software (Eclat) has achieved a classification accuracy of 93.1% on a test set of 3217 EST sequences from Hordeum vulgare and Blumeria graminis, which is a significant improvement compared to PF-IND (prediction accuracy of 81.2% on the same test set). EST sequences with at least 50 nt of coding sequence can be classified using Eclat with high confidence. Eclat allows training of classifiers for any host-pathogen combination for which there are sufficient classified training sequences. AVAILABILITY: Eclat is freely available on the Internet (http://mips.gsf.de/proj/est) or on request as a standalone version. CONTACT: friedel@informatik.uni-muenchen.de.     

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein‐Barr virus orthologs

Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein‐Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX‐TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine‐rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co‐ordinate nuclear and cytoplasmic processes.      

Development of accessory reproductive glands and its control by the corpus allatum in adult male Melanoplus sanguinipes

Changes in the protein content of and the rate at which labelled protein appears in the accessory reproductive glands (ARG), fat body, and haemolymph were studied in normal and allatectomized (CA^?) males of the migratory grasshopper, Melanoplus sanguinipes. In addition, the effects of treatment of CA^? insects with synthetic juvenile hormone (SJH), copulation, and removal of the ARG were examined.In normal males the protein content of the ARG increases linearly during the first 14 days after emergence. Incorporation of label by the ARG is maximal at day 7 and then decreases until, at day 14, it is the same as at day 1. The protein content of the fat body and haemolymph increases up to day 10 then declines, whereas changes in the uptake of label by the fat body and haemolymph parallel those of the ARG.Removal of the corpora allata (CA) prevents the normal increase in protein content of the ARG, but the protein content of the fat body and haemolymph increases steadily throughout the 14 days. Incorporation of label into the ARG, fat body, and haemolymph remained low throughout the experiment. Treatment of CA^? insects with SJH, or copulation, stimulates the uptake of label by the ARG, fat body, and haemolymph and also results in an increase in their protein content.Removal of the ARG leads to an increase in the protein content of the fat body and haemolymph. Uptake of label by the fat body remains low after the operation. Although the rate at which labelled protein appears in the haemolymph is high initially, it declines steadily to day 14.We conclude that the CA regulate ARG development. It is suggested that the fat body, under CA control, synthesizes proteins which are incorporated into secretions of the ARG. Further, it is proposed that the primary effect of copulation is activation of the CA.     

Common patterns in type II restriction enzyme binding sites

Restriction enzymes are among the best studied examples of DNA binding proteins. In order to find general patterns in DNA recognition sites, which may reflect important properties of protein–DNA interaction, we analyse the binding sites of all known type II restriction endonucleases. We find a significantly enhanced GC content and discuss three explanations for this phenomenon. Moreover, we study patterns of nucleotide order in recognition sites. Our analysis reveals a striking accumulation of adjacent purines (R) or pyrimidines (Y). We discuss three possible reasons: RR/YY dinucleotides are characterized by (i) stronger H-bond donor and acceptor clusters, (ii) specific geometrical properties and (iii) a low stacking energy. These features make RR/YY steps particularly accessible for specific protein–DNA interactions. Finally, we show that the recognition sites of type II restriction enzymes are underrepresented in host genomes and in phage genomes.