Decoding temporally encoded sensory input by cortical oscillations and thalamic phase comparators

The temporally encoded information obtained by vibrissal touch could be decoded “passively,” involving only input-driven elements, or “actively,” utilizing intrinsically driven oscillators. A previous study suggested that the trigeminal somatosensory system of rats does not obey the bottom-up order of activation predicted by passive decoding. Thus, we have tested whether this system obeys the predictions of active decoding. We have studied cortical single units in the somatosensory cortices of anesthetized rats and guinea pigs and found that about a quarter of them exhibit clear spontaneous oscillations, many of them around whisking frequencies (≈10 Hz). The frequencies of these oscillations could be controlled locally by glutamate. These oscillations could be forced to track the frequency of induced rhythmic whisker movements at a stable, frequency-dependent, phase difference. During these stimulations, the response intensities of multiunits at the thalamic recipient layers of the cortex decreased, and their latencies increased, with increasing input frequency. These observations are consistent with thalamocortical loops implementing phase-locked loops, circuits that are most efficient in decoding temporally encoded information like that obtained by active vibrissal touch. According to this model, and consistent with our results, populations of thalamic “relay” neurons function as phase “comparators” that compare cortical timing expectations with the actual input timing and represent the difference by their population output rate.

Participation of the nuclear cap binding complex in pre-mRNA 3′ processing

Communication between the 5′ and 3′ ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5′ end is bound by the nuclear cap binding complex (CBC). This RNA–protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3′ end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5′ and 3′ ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3′ processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of “closed-loops,” due to interactions between factors bound at their 5′ and 3′ ends.

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes

The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions.

SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase

Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by Salmonella dublin, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro. SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca2+-dependent chloride secretion. In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate to yield inositol 1,4,5,6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the inhibition of chloride secretion by phosphatidylinositol 3,4,5-trisphosphate [Eckmann, L., Rudolf, M. T., Ptasznik, A., Schultz, C., Jiang, T., Wolfson, N., Tsien, R., Fierer, J., Shears, S. B., Kagnoff, M. F., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14456–14460]. Mutation of a conserved cysteine that abolishes phosphatase activity of SopB results in a mutant strain, S. dublin SB c/s, with decreased ability to induce fluid secretion in infected calf intestine loops. Moreover, HeLa cells infected with S. dublin SB c/s do not accumulate high levels of inositol 1,4,5,6-tetrakisphosphate that are characteristic of wild-type S. dublin-infected cells. Therefore, SopB mediates virulence by interdicting inositol phosphate signaling pathways.

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells

An Fcα receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice. Paired Ig-like receptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcαR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only one PIR-B gene and multiple PIR-A genes. The PIR-A and PIR-B genes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conserved PIR-A and PIR-B genes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses.

λ Rap protein is a structure-specific endonuclease involved in phage recombination

Bacteriophage λ encodes a number of genes involved in the recombinational repair of DNA double-strand breaks. The product of one of these genes, rap, has been purified. Truncated Rap proteins that copurify with the full-length form are derived, at least in part, from a ρ-dependent transcription terminator located within its coding sequence. Full-length and certain truncated Rap polypeptides bind preferentially to branched DNA substrates, including synthetic Holliday junctions and D-loops. In the presence of manganese ions, Rap acts as an endonuclease that cleaves at the branch point of Holliday and D-loop substrates. It shows no obvious sequence preference or symmetry of cleavage on a Holliday junction. The biochemical analysis of Rap gives an insight into how recombinants could be generated by the nicking of a D-loop without the formation of a classical Holliday junction.

Leading role of thalamic over cortical neurons during postinhibitory rebound excitation

The postinhibitory rebound excitation is an intrinsic property of thalamic and cortical neurons that is implicated in a variety of normal and abnormal operations of neuronal networks, such as slow or fast brain rhythms during different states of vigilance as well as seizures. We used dual simultaneous intracellular recordings of thalamocortical neurons from the ventrolateral nucleus and neurons from the motor cortex, together with thalamic and cortical field potentials, to investigate the temporal relations between thalamic and cortical events during the rebound excitation that follows prolonged periods of stimulus-induced inhibition. Invariably, the rebound spike-bursts in thalamocortical cells occurred before the rebound depolarization in cortical neurons and preceded the peak of the depth-negative, rebound field potential in cortical areas. Also, the inhibitory-rebound sequences were more pronounced and prolonged in cortical neurons when elicited by thalamic stimuli, compared with cortical stimuli. The role of thalamocortical loops in the rebound excitation of cortical neurons was shown further by the absence of rebound activity in isolated cortical slabs. However, whereas thalamocortical neurons remained hyperpolarized after rebound excitation, because of the prolonged spike-bursts in inhibitory thalamic reticular neurons, the rebound depolarization in cortical neurons was prolonged, suggesting the role of intracortical excitatory circuits in this sustained activity. The role of intrathalamic events in triggering rebound cortical activity should be taken into consideration when analyzing information processes at the cortical level; at each step, corticothalamic volleys can set into action thalamic inhibitory neurons, leading to rebound spike-bursts that are transferred back to the cortex, thus modifying cortical activities.

Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells

Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.

A very limited number of keywords (main patterns) describes all sequences of the human variable heavy (VH) and κ (Vκ) domains

Sequences of the variable heavy (VH) and κ (Vκ) domains of Ig structures were divided into 21 fragments that correspond to strands, loops, or parts of these structural units of the variable domains. Amino acid sequences of fragments (termed “words”) were collected from the 1,172 human heavy and 668 human κ chains available in the Kabat database. Statistical analysis of words of 17 fragments was performed (fragments that comprise the complementary determining regions′ fragments will not be discussed in this paper). The number of different words (those with different residues in at least one position) ranged, for various fragments, from 11 to 75 in the κ chains, and from 23 to 189 in the heavy chains. The main result of this study is that very few keywords, or main patterns of words, were necessary to describe over 90% of the sequences (no more than two keywords per fragment in the κ and no more than five per fragment in the heavy chains). No identical keywords were found for different fragments of the variable domains. Keywords of aligned fragments of the VH and Vκ domains were different in all but two instances. Thus, knowing the keywords, one can determine whether any given small part of a sequence belongs to a heavy or κ chain and predict its precise localization in the sequence. In addition, by using all of the keywords obtained through analysis of the Kabat database, it was possible to describe completely the sequences of the human VH and Vκ germ-line segments.

The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

Assembly of Lampbrush Chromosomes from Sperm Chromatin

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24–48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific “landmarks.” Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.