Unique Tail Research Articles

Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin

Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with <45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil. Like several other unconventional myosins and a plant kinesin, myosin-X contains both a myosin tail homology 4 (MyTH4) domain and a FERM (band 4.1/ezrin/radixin/moesin) domain. The unique tail domain also includes three pleckstrin homology domains, which have been implicated in phosphatidylinositol phospholipid signaling, and three PEST sites, which may allow cleavage of the myosin tail. Most intriguingly, myosin-X in cultured cells is present at the edges of lamellipodia, membrane ruffles, and the tips of filopodial actin bundles. The tail domain structure, biochemical features, and localization of myosin-X suggest that this novel unconventional myosin plays a role in regions of dynamic actin.

KRN5500 induces apoptosis (PCD) of myeloid leukemia cell lines and patient blasts

The purpose of this study was to determine if KRN5500, a spicamycin derivative with a unique acyl tail, would induce programmed cell death (PCD) of myeloid leukemia cell lines and cryopreserved leukemic blasts from newly diagnosed children with acute leukemia (AL). Cells were incubated with varying concentrations (0–5 ng/ml) of KRN5500 and the percent PCD determined using a modified in situ end labeling (ISEL) technique with Klenow fragment. The percent PCD was calculated using the formula: Percent PCD (% PCD)=[number of apoptotic cells/(viable cells+apoptotic cells)]×100. DMSO (0.30% w/v) was added to the cells in culture as the positive control for PCD; the negative control was media or albumin. KRN5500 increased the amount of PCD significantly in all five of the tested cell lines; U937 41±1.8%, KG1a 40±0.3%, HEL 14±2.2%, HL-60 41±0.9%, K562 36±2% (mean PCD±SD). Patient blasts exposed to KRN5500 had an increase in PCD when exposed to 2 ng/ml of agent from 2 to 8 h; acute myeloid leukemia patients 7.5±0.5% at 2 h to 43.5±1.6% at 8 h, and acute lymphocytic leukemia patients rose from 12.4±3.8% at 2 h to 29.9±11.6% after 8 h (mean±SE). Overall the PCD for the patient samples was 3.7 versus 28±4% at 2 and 8 h, respectively. PCD was proportional to the dose of KRN5500 and incubation time. Further pre-clinical and clinical studies are required.

Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1). Organization of the approximately 400 kDa subunit, and amino acid sequence of its functional units f, g and h.

We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abalone Haliotis tuberculata. HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a approximately 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well-studied hemocyanin types of the closely related marine gastropod Megathura crenulata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, SDS/PAGE and N-terminal sequencing, we identified eight different 40-60 kDa functional units in HtH1, termed HtH1-a to HtH1-h, and determined their linear arrangement within the elongated subunit. From Haliotis mantle tissue, rich in hemocyanin-producing pore cells, we isolated mRNA and constructed a cDNA library. By expression screening with HtH-specific rabbit antibodies, a cDNA clone was isolated and sequenced which codes for the three C-terminal functional units f, g and h of HtH1. Their sequences were aligned to those available from other molluscs, notably to functional unit f and functional unit g from the cephalopod Octopus dofleini. HtH1-f, which is the first sequenced functional unit of type f from a gastropod hemocyanin, corresponds to functional unit f from Octopus. Also functional unit g from Haliotis and Octopus correspond to each other. HtH1-h is a gastropod hemocyanin functional unit type which is absent in cephalopods and has not been sequenced previously. It exhibits a unique tail extension of approximately 95 amino acids, which is lacking in functional units a to g and aligns with a published peptide sequence of 48 amino acids from functional unit h of Helix pomatia hemocyanin. The new Haliotis sequences are discussed with respect to their counterparts in Octopus, the 15 A three-dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 A X-ray structure of functional unit g from Octopus hemocyanin.

Cloning and Characterization of a Novel RING Finger Protein That Interacts with Class V Myosins

We have identified a novel protein (BERP) that is a specific partner for the tail domain of myosin V. Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles. BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain. A yeast two-hybrid screening indicated that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb). This interaction was confirmed by immunoprecipitation, which also demonstrated that BERP could associate with myosin Va, the product of the dilute gene. Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells. We also found that the RBCC domain of BERP is involved in protein dimerization. Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain resulted in defective PC12 cell spreading and prevented neurite outgrowth in response to nerve growth factor. Our studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport.

Characterization of the Ca2+-dependent binding of annexin IV to surfactant protein A

We have shown previously that surfactant protein A (SP-A) binds to annexin IV in a Ca2+-dependent manner [Sohma, Matsushima, Watanabe, Hattori, Kuroki and Akino (1995) Biochem. J. 312, 175-181]. Annexin IV is a member of the annexin family having four consensus repeats of about 70 amino acids and a unique N-terminal tail. In the present study, the functional site of both annexin IV and SP-A for the Ca2+-dependent binding was investigated using mutant proteins. SP-A bound in a Ca2+-dependent manner to an annexin-IV truncation mutant consisting of the N-terminal domain and the first three domains (T(N-1-2-3)). SP-A also bound to T3-4, but this interaction was not Ca2+-dependent. SP-A bound weakly to the other truncation mutants (T(N-1-2), T(2-3) and T(2-3-4)). Each consensus repeat of annexin IV possesses a conserved acidic amino acid residue (Glu70, Asp142, Glu226 and Asp301) that putatively ligates Ca2+. Using annexin-IV DE mutants in which one, two or three residues out of the four Asp/Glu were altered to Ala by site-directed mutagenesis [Nelson and Creutz (1995) Biochemistry 34, 3121-3132], it was revealed that Ca2+ binding in the third domain is more important than in the other Ca2+-binding sites. SP-A is a member of the animal lectin group homologous with mannose-binding protein A. The substitution of Arg197 of rat SP-A with Asp or Asn eliminated binding to annexin IV, whereas the substitution of Glu195 with Gln was silent. These results suggest that the Ca2+ binding to domain 3 of annexin IV is required for the Ca2+-dependent binding by SP-A and that Arg197 of SP-A is important in this binding.

A unique isoform of phospholipase Cβ4 highly expressed in the cerebellum and eye

We report a unique isoform of PLCβ4 in rat, PLCβ4c, that has an additional 37-nucleotide exon inserted between nucleotides 3459–3460 of the previously published PLCβ4a coding sequence. This insertion results in replacement of 22 amino acid residues at the carboxyl terminal tail of PLCβ4a with 41 unique residues. A human EST for PLCβ4 also contains this exon and this exon was mapped to within a 5.5 kb intron of the human PLCβ4 gene. PLCβ4c is the third PLCβ4 isoform to be identified which has a unique carboxyl-terminal tail. PLCβ4b differs from PLCβ4a by truncation 162 amino acid residues from the carboxyl terminus which are replaced with 10 distinct amino acid residues. Reverse transcription–polymerase chain reaction experiments show that both PLCβ4a and PLCβ4c mRNA are expressed throughout the rat brain and that PLCβ4c mRNA is highly expressed in the eye and cerebellum. RNase protection assays demonstrate that both PLCβ4a and PLCβ4c transcripts are abundant in the cerebellum. The different carboxyl terminal tails of PLCβ4 isoforms may allow for differential targeting and subcellular localization, contributing to regulation of PLC β4-mediated signal transduction.

Directed expression of keratin 16 to the progenitor basal cells of transgenic mouse skin delays skin maturation.

We previously hypothesized that the type I keratin 16 (K16) plays a role in the process of keratinocyte activation that occurs in response to skin injury (Paladini, R.D., K. Takahashi, N.S. Bravo, and P.A. Coulombe. 1996. J. Cell Biol. 132:381-397). To further examine its properties in vivo, the human K16 cDNA was constitutively expressed in the progenitor basal layer of transgenic mouse skin using the K14 gene promoter. Mice that express approximately as much K16 protein as endogenous K14 display a dramatic postnatal phenotype that consists of skin that is hyperkeratotic, scaly, and essentially devoid of fur. Histologically, the epidermis is thickened because of hyperproliferation of transgenic basal cells, whereas the hair follicles are decreased in number, poorly developed, and hypoproliferative. Microscopically, the transgenic keratinocytes are hypertrophic and feature an altered keratin filament network and decreased cell-cell adhesion. The phenotype normalizes at approximately 5 wk after birth. In contrast, control mice expressing a K16-K14 chimeric protein to comparable levels are normal. The character and temporal evolution of the phenotype in the K16 transgenic mice are reminiscent of the activated EGF receptor- mediated signaling pathway in skin. In fact, tyrosine phosphorylation of the EGF receptor is increased in the newborn skin of K16 transgenic mice. We conclude that expression of K16 can significantly alter the response of skin keratinocytes to signaling cues, a distinctive property likely resulting from its unique COOH-terminal tail domain.

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I–IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together, with the finding that the yeast DNA ligase IV homologue is essential for non-homologous DNA end joining [5–7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein–protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].

Core domain mutation (S86Y) selectively inactivates polyubiquitin chain synthesis catalyzed by E2-25K.

The mammalian ubiquitin conjugating enzyme known as E2-25K catalyzes the synthesis of polyubiquitin chains linked exclusively through K48-G76 isopeptide bonds. The properties of truncated and chimeric forms of E2-25K suggest that the polyubiquitin chain synthesis activity of this E2 depends on specific interactions between its conserved 150-residue core domain and its unique 50-residue tail domain [Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537]. In the present study, we provide strong support for this model by showing that a point mutation in the core domain (S86Y) mimics the effect of deleting the entire tail domain: the ability to form an E2 approximately ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (>/=100-fold reduction in kcat/Km). The properties of E2-25K enzymes carrying the S86Y mutation indicate that this mutation strengthens the interaction between the core and tail domains: both free and ubiquitin-bound forms of S86Y-25K are completely resistant to tryptic cleavage at K164 in the tail domain, whereas wild-type enzyme is rapidly cleaved at this site. Other properties of S86Y-26K suggest that the active site of this mutant enzyme is more occluded than the active site of the wild-type enzyme. (1) Free S86Y-25K is alkylated by iodoacetamide 2-fold more slowly than the wild-type enzyme. (2) In assays of E2 approximately ubiquitin thiol ester formation, S86Y-25K shows a 4-fold reduced affinity for E1. (3) The ubiquitin thiol ester adduct of S86Y-25K undergoes (uncatalyzed) reaction with dithiothreitol 3-fold more slowly than the wild-type thiol ester adduct. One model to accommodate these findings postulates that an enhanced interaction between the core and tail domains, induced by the S86Y mutation, causes a steric blockade at the active site which prevents access of the incoming ubiquitin acceptor to the thiol ester bond. Consistent with this model, the S86Y mutation inhibits ubiquitin transfer to macromolecular acceptors (ubiquitin and polylysine) more strongly than transfer to small-molecule acceptors (free lysine and short peptides). These results suggest that unique residues proximal to E2 active sites may influence specific function by mediating intramolecular interactions.

Unique Sets Oriented Parallelization of Loops with Non-uniform Dependences

Although many methods exist for nested loop partitioning, most of them perform poorly when parallelizing loops with non-uniform dependences. This paper addresses the issue of automatic parallelization of loops with non-uniform dependences. Such loops are normally not parallelized by existing parallelizing compilers and transformations. Even when parallelized in rare instances, the performance is very poor. Our approach is based on the 'convex hull' theory which has adequate information to handle non-uniform dependences. We introduce the concept of'complete dependence convex hull', 'unique head and tail sets' and abstract the dependence information into these sets. These sets form the basis of the iteration space partitions. The properties of the unique head and tail sets are derived. Depending on the relative placement of these unique sets, partitioning schemes are suggested for implementation of our technique. Implementation results of our scheme on the Cray J916 and comparison with other schemes show the superiority of our technique.

Epidermal growth factor induces glucose storage in transgenic 3T3-L1 adipocytes overexpressing epidermal growth factor receptors.

3T3-L1 adipocytes represent an established physiological model for studying glucose uptake and storage. Overexpression of epidermal growth factor (EGF) receptors in these cells (200,000-250,000 receptors per cell) confers EGF-inducible GLUT4-mediated glucose uptake (17). We now report that EGF receptor (EGFR)-mediated signals can induce incorporation of glucose into glycogen and lipids in these cells. Incorporation into lipids was stimulated to similar levels by insulin or EGF in adipocytes expressing full-length (wild type) EGFR (2.05 +/- 0.26-fold for insulin vs. 2.28 +/- 0.15-fold for EGF). EGF induced incorporation into glycogen at roughly 60% of the level of insulin (4.53 +/- 0.57-fold for insulin vs. 2.76 +/- 0.25-fold for EGF); this corresponded with similarly lower levels of glycogen synthase activation by EGF relative to insulin stimulation. EGFR kinase activity was required for induced storage because a kinase-inactive (M721) EGFR failed to stimulate glucose incorporation into glycogen or lipids. EGFRs that lack all or part of the unique EGFR COOH-terminal tail induced glucose incorporation at levels similar to that stimulated by full-length (wild type) EGFR. Thus, domains in the COOH-terminal tail of the EGFR, which are necessary for stimulating glucose transport, are not required for signaling EGF-induced glucose storage. EGF-induced glucose storage did not require de novo protein synthesis, suggesting that EGFR signaling uses existing pathways in the adipocytes. These data demonstrate that signaling pathways for EGFR-mediated glucose storage and GLUT4-mediated glucose transport diverge at the receptor level. Thus, EGF-induced glucose storage can be achieved in the absence of induced GLUT4-mediated glucose transport.

Display Behavior and Natural History of the Yellow-Crowned Manakin (Heterocercus flavivertex: Pipridae)

The behavior and natural history of the Yellow-crowned Manakin (Heterocercus flavivertex, Pipridae) were observed on the Rio Ventuari in northern Territorio Federal Amazonas, Venezuela. Male flavivertex have a solitary lek breeding system, a display repertoire including five distinct vocalizations, several display elements, and a probable mechanical display sound. The tail-shiver display, in which the cocked tail is prominently featured, has not been observed in other Heterocercus species, but this display element may be an explanation for the evolution of the unique tail shape in the genus. Other flavivertex display elements, such as the above-the-canopy display flight, are behavioral homologs shared with other Heterocercus species. Female H. flavivertex construct hanging cup nests that are similar in architecture, composition, and placement to other manakin species. The diet of H. flavivertex is apparently composed of a greater quantity of insects than other manakin species. The evolution of display, morphology, diet, and social behavior in Heterocercus is discussed.

Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.

Isoforms of the EP3 subtype of human prostaglandin E2 receptor transduce both intracellular calcium and cAMP signals.

The EP3 subtype of prostaglandin E2 receptor transduces diverse physiological responses in mammalian tissues through signaling pathways coupled to heterotrimeric G proteins. Distinct cDNA clones encoding five isoforms of the EP3 receptor were isolated from a human uterus cDNA library. The human EP3 receptor isoforms designated hEP3-I, I', II, III, and IV are derived from alternative RNA splicing and differ only in the distal sequences of their carboxyl-terminal cytoplasmic tails. The unique cytoplasmic tails consist of 31 amino acids for isoforms I and I', 29 for II, 6 for III, and 15 for IV. When stably expressed in CHO cell transfectants, all isoforms exhibited similar EP3-specific binding of [3H]-PGE2 and PGE2 analogs. The EP3-selective agonist M&B 28767 both decreased the intracellular cAMP concentration ([cAMP]i) and increased the intracellular concentration of calcium ([Ca2+]i) with quantitative differences among different isoforms, but none mediated an increase in [cAMP]i. Pertussis toxin treatment completely blocked the decrease in [cAMP]i, but not the increase in [Ca2+]i evoked by M&B 28767. PGE2-induced desensitization of [3H]PGE2 binding by isoforms III and IV was rapid and transient, whereas that by isoform II was slow and persistent. Reverse transcription-PCR amplification of EP3 receptor messages in human kidney and uterine tissue RNA detected expression of all isoforms with different abundancies. The dual signal transduction pathways and distinctive tissue distribution of isoforms of the EP3 receptor are consistent with its mediation of diverse functions of PGE2.

Ultrastructural colocalization of growth hormone binding protein and pituitary hormones in adenohypophyseal cells of the rat.

GH receptor immunoreactivity is widely distributed within the rat pituitary gland, although apart from somatotrophs the cell types with GH receptor immunoreactivity have yet to be identified. It is also unknown whether this immunoreactivity reflects the presence of GH binding proteins (GHBPs) or authentic receptors. The possible colocalization of GHBPs and pituitary hormones in somatotrophs, lactotrophs, gonadotrophs, thyrotrophs, and corticotrophs was therefore examined using immunogold electron microscopy. Pituitary sections were indirectly immunostained with antibodies for GH, PRL, LH, FSH, TSH, or ACTH using gold-labeled immunoglobulin G. The same grids were also immunostained with a polyclonal antibody raised against rat GHBP using protein A conjugated to gold particles of a different size. Some sections were gold-labeled using a monoclonal antibody (MAb 4.3) raised against the unique hydrophobic tail of the GHBP, using gold-labeled immunoglobulin G. The cellular and ultrastructural distribution of immunoreactivity within the pituitary gland was similar after labeling with either GHBP antiserum. In all immunoreactive cells the labeling was most intense in secretory granules. Specific staining was not, however, demonstrated in the nucleus, in contrast with earlier findings using MAb 263. Moreover, while staining with MAb 263 appeared to be ubiquitous, some pituitary cells were not labeled by the polyclonal GHBP antiserum or by MAb 4.3. GHBP immunoreactivity was, however, colocalized with hormones in all pituitary cell-types, although in some cells GHBP immunoreactivity was not present in all secretory granules. These results clearly demonstrate the presence of GHBPs in adenohypophyseal cells, in which they appear to be stored or secreted with GH, PRL, LH, FSH, TSH, and ACTH. The function of GHBPs in the pituitary gland is, however, uncertain.

The largest subunit of RNA polymerase II in Dictyostelium: conservation of the unique tail domain and gene expression.

cDNAs encoding the largest subunit of RNA polymerase II were isolated from a Dictyostelium cDNA library. A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced. Similar to other eukaryotic RNA polymerases II, the largest subunit of Dictyostelium RNA polymerase II contains a unique repetitive tail domain at its carboxyl-terminal region. It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein. RNA blots show that cDNA probes hybridized with a single mRNA species of approximately 6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons. Interestingly, expression of the largest subunit of RNA polymerase II appears to be under developmental regulation. The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h. Cells began to accumulate a higher level of the RNA polymerase II mRNA after 9 h of development. When cells were treated with low concentrations of cAMP pulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells.

Identification and developmental expression of a novel low molecular weight neuronal intermediate filament protein expressed in Xenopus laevis

Xenopus laevis is a valuable model system for the study of vertebrate neuroembryogenesis. However, very few well-characterized nervous system-specific molecular markers are available for studies in this organism. We screened a X. laevis adult brain cDNA library using a cDNA probe for mouse low molecular weight neurofilament protein (NF-L) in order to identify neuron-specific intermediate filament proteins. Clones for two distinct neuron-specific intermediate filament proteins were isolated and sequenced. One of these encoded for a Xenopus NF-L (XNF-L) and the other for a novel neuron-specific Xenopus intermediate filament protein (XNIF) that was present earlier and more abundantly than XNF-L during development. XNIF contained a central rod domain with multiple sequence features characteristic of IF proteins. The XNF-L was very similar to mouse NF-L, with a 77% sequence identity in the rod domain and the presence of a polyglutamic acid region in the tail domain, characteristic of type IV neurofilament proteins. In contrast, XNIF showed only 60% identity to mouse NF-L in the rod domain and lacked the glutamic acid-rich sequence in the tail domain. XNIF also had a very low (approximately 38%) sequence identity in the head and tail domains as compared to NF-L and other neurofilament proteins (45% identity to the head domain of alpha-internexin). In the adult frog, XNIF mRNA is detected by Northern blots only within the nervous system and by in situ hybridization histochemistry exclusively in neurons, particularly in the medullary reticular system and spinal cord. Antisera raised against the unique tail region of XNIF detected a single distinct 60 kDa band in Western blots of nervous system cytoskeletal preparations, and this XNIF immunoreactivity was concentrated in axons in the PNS and in small perikarya in the dorsal root ganglion. In contrast, NF-L immunoreactivity was principally in the large perikarya in the dorsal root ganglion. In development, XNIF mRNA appears more abundant than XNF-L mRNA in all premetamorphic stages examined. XNIF mRNA is first detectable at stage 24 (26 hr), whereas stable expression of XNF-L is at stage 35/36 (50 hr). XNIF immunoreactivity is detectable within the cement gland, within many neuronal cell bodies and axon tracts within the developing nervous system, and within all cellular layers of the developing retina. The availability of these two distinct neuron-specific intermediate filament proteins, with different temporal and spatial expression patterns, should provide new markers as well as targets for functional perturbation in the developing X. laevis nervous system.

Identification of a novel growth hormone binding protein mRNA in rat liver

The presence of highly specific serum binding proteins for growth hormone (GH) has been well characterized in many species. In the rat the major growth hormone-binding protein (GH-BP) is a truncated, variant form of the target tissue GH receptor and is derived by an alternative mRNA splicing event. The GHBP mRNA is coexpressed in all tissues expressing the full length GH receptor. In the present study, we have made an oligonucleotide probe to the unique hydrophilic tail of the rat GHBP mRNA (1.2kb) and identified a novel GHBP-like mRNA of 2.6 kb transcript in addition to the 1.2 kb transcript. This unique 2.6 kb transcript was expressed/detected only in rat liver. There was no significant difference in abundance between the sexes or during pregnancy, implying that this transcript may be regulated independently of the 1.2 kb mRNA. The 2.6 kb transcript was clearly identifiable in the fetus, as was the 1.2 kb transcript, but showed virtually no change in abundance with age, in sharp contrast to the 1.2 kb mRNA, which has a distinct developmental pattern, being low in the fetus and peaking early postnatally. RNAse H treatment suggested that this 2.6 kb transcript is polyadenylated. A corresponding 2.6 kb mRNA has been detected using a longer cDNA or cRNA probe for the GH-binding domain of the rat GHR/GHBP. These data collectively suggest that the 2.6 kb mRNA transcript is a bonafide but tissue-specific GHBP mRNA and that the 1.2 and 2.6 kb mRNAs are likely to differ primarily with respect to the length of the 3' untranslated region of the sequence.

A tale of two motors

Eukaryotic molecular motors constitute two families, one actin-based and the other microtubule-based. More than 25 members of these two families have been identified. Primary structure reveals that each member consists of a conserved motor unit attached to a unique tail. The tail probably directs the cellular localization of the motor and regulates its function.

A new Acanthamoeba myosin heavy chain. Cloning of the gene and immunological identification of the polypeptide

An Acanthamoeba myosin heavy chain has been identified whose tail domain amino acid sequence distinguishes it from Acanthamoeba myosins IB, IC, and II. The gene for this novel myosin heavy chain spans approximately 6.8 kilobases, is split by 17 introns, and encodes a 177-kDa polypeptide. While the amino-terminal approximately 90 kDa of this polypeptide is highly similar to the globular head sequences of myosins I and II, its approximately 87-kDa tail domain shows essentially no similarity to the tail sequences of either type of myosin. The only exception to this is the carboxyl-terminal approximately 50-amino acid region of the polypeptide, which is homologous to the carboxyl termini of the myosins I. Interestingly, this approximately 50-residue segment has been shown to exist in a diverse family of cytoskeleton-associated proteins that include nonreceptor tyrosine kinases, phospholipase C gamma, and fodrin (Rodaway, A. R. F., Sternberg, M. J. E., and Bentley, D. L. (1989) Nature 342, 624). Sequence analysis indicates that the tail domain of this new myosin is incapable of forming a myosin II-like coiled-coil structure, implying that the protein is single-headed and nonfilamentous. For this reason we have tentatively classified it as a high molecular weight form of myosin I (HMWMI). To determine if HMWMI exists in cells, antiserum was raised against a bacterially expressed fusion peptide made using a cDNA clone encoding most of the unique HMWMI tail domain. This antiserum does not recognize Acanthamoeba myosins IB, IC, or II but does recognize a single polypeptide in whole cell extracts with the mobility predicted for the HMWMI heavy chain. This protein is precipitated from crude extracts using F-actin and released from the pellet by ATP, supporting its classification as a member of the myosin family of proteins.