Multiple nuclear localization sequences in SRSF4 protein

SRSF4 is one of the members of serine‐/arginine (SR)‐rich protein family involved in both constitutive and alternative splicing. SRSF4 is localized in the nucleus with speckled pattern, but its nuclear localization signal was not determined. Here, we have identified nuclear localization signals (NLSs) of SRSF4 by using a pyruvate kinase fusion system. As expected, arginine‐/serine (RS)‐rich domain of SRSF4 confers nuclear localization activity when it is fused to PK protein. We then further delineated the minimum sequences for nuclear localization in RS domain of SRSF4. Surprisingly, RS‐rich region does not always have a nuclear localization activity. In addition, basic amino acid stretches that resemble to classical‐type NLSs were identified. These results strongly suggest that SRSF4 protein uses two different nuclear import pathways with multiple NLSs in RS domain.     

Identification of the nuclear localization signal of human papillomavirus type 16 L1 protein.

Human papillomavirus type 16(HPV16) L1 and L2 capsid proteins can be detected only in the nucleus of infected cells. For other nuclear proteins, specific sequences of basic amino acids(aa) termed nuclear localization signals (NLS) direct the protein from the cytoplasm to the nucleus. We used a series of deletion and substitution mutations of the HPV16 L1 protein, produced by recombinant vaccinia virus (rVV), to identify NLS within HPV16 L1 and showed that HPV16 L1 contains two NLS sequences, each containing basic aa clusters. One NLS consisted of 6 basic amino acids (KRKKRK from aa 525 to 530) at the carboxy terminal end of L1. The other NLS contained 2 basic aa clusters(KRK from aa 510 to 512 and KR at aa 525, 526) separated by 12 amino acids. Mutations in either NLS did not alter nuclear localization of L1 when the other remained intact, but mutations to both prevented nuclear localization of L1. The L1 NLS could be overridden by introduction of a membrane binding sequence at the amino terminal end of the protein. A databases search showed that all sequenced papillomaviruses are predicted to have L1 and L2 capsid proteins with sequences of basic amino acids homologous with one or both NLS of HPV16 L1.     

Herpes virus-like sequences are specifically found in Kaposi sarcoma lesions.

AIM: To detect the prevalence of herpes virus-like DNA sequences in AIDS associated Kaposi sarcoma (KSHV) lesions and normal tissue. METHODS: KSHV detection was performed by polymerase chain reaction (PCR) using four different sets of primers. PCR products were cloned, sequenced, and analysed. RESULTS: All of four biopsies of Kaposi sarcoma lesions and all of three paraffin embedded Kaposi sarcoma tissues were positive for KSHV, while normal tissue from the same patients was negative. Sequence analysis of amplification products revealed polymorphisms that result in amino acid changes of the predicted sequence. CONCLUSIONS: KSHV is prevalent in tissues from Kaposi sarcoma, suggesting a role in the development of the tumour. On this basis, anti-herpes virus agents should be considered to control Kaposi sarcoma.     

Identification of alternatively spliced human biotinidase mRNAs and putative localization of endogenous biotinidase

Biotinidase is essential for recycling the vitamin biotin and for transferring biotin to proteins, such as histones, suggesting that the enzyme localizes to various cellular and extracellular sites. To better understand the functions of the enzyme, we examined its gene structure and subcellular localization. Using RACE-PCR and a BLAST search, we extended the 5' sequence of the biotinidase gene. Three novel, alternatively spliced variants of biotinidase, 1a, 1b, and 1c, were identified in multiple human tissues. Exon 1c is present only in testes. The sequence of the 5' splice variants, 1a and 1b, suggest that biotinidase localizes to the mitochondria and/or ER, respectively. Using indirect immunofluorescence studies, biotinidase localizes to organelles in the cytoplasm, but not nucleus, of human fibroblasts and Hep G2 cells. Endogenous expression was examined by isopycnic gradient centrifugation of rat liver organelles, which identified an 85kDa biotinidase protein with biotinyl-hydrolase and transferase activities in microsomes and possibly lysosomes. A 48kDa protein, which also reacts with anti-biotinidase, localizes to mitochondria. The 48kDa protein is not N-glycosylated but is biotinylated, is in the inner mitochondrial matrix, but has no biotinyl-hydrolase or transferase activities. The function and validation of the mitochondrial species remains to be determined. The 5' splice variants and organelle fractionation studies indicate that biotinidase is directed to the secretory pathway and perhaps mitochondria.     

Regional localization of 3 Y-derived sequences on the human X and Y chromosomes

The structure of the Y chromosome and the relationship between the human sex chromosomes have been studied using Y-derived sequences cloned in cosmids. Two probes recognize different unique sequences which map to the heterochromatic part of the long arm of the Y chromosome. A third sequence is shared by the long arm of the X chromosome and the euchromatic part of the Y chromosome. Thus homology between the sex chromosomes occurs outside the pairing region.     

Immunocytochemical localization of putative cholinergic neurons in the goldfish retina

The presence of putative cholinergic neurons in goldfish retina was demonstrated by immunocytochemical localization of choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Four populations of ChAT-immunoreactive neurons were localized: two with cell bodies in the inner nuclear layer and two with cell bodies in the ganglion cell layer. The processes of these neurons ramified in lamina 2 and/or 4 (of 5) in the inner plexiform layer. These cell populations are comparable to populations of putative cholinergic neurons that have been identified by [3H]choline uptake [3, 10].     

Anti-complement immunofluorescence establishes nuclear localization of human cytomegalovirus matrix protein

A monospecific, polyclonal antiserum to the 69-kDa matrix protein of human cytomegalovirus (HCMV) was prepared in a guinea pig and used to determine the intracellular distribution of this viral antigen. The resulting antiserum was specific for infected cells as tested by immunofluorescence, and specific for the HCMV matrix protein as determined by "nitrocellulose immunoassay" of electrophoretically separated, infected-cell proteins. Antibodies were reacted with fixed, infected human fibroblasts, and visualized by the anti-complement immunofluorescence procedure to avoid complications arising from the strong IgG Fc binding activity of the infected-cell-specific cytoplasmic inclusion. Results establish that the matrix protein is located in the nucleus, and indicate that it is concentrated in the nucleoplasm rather than within the intranuclear inclusions.     

Characterization of two human monoclonal IgM antibodies that recognize nuclear lamins.

Using immunofluorescence and immunoblotting techniques, we have identified monoclonal IgM lambda from two patients that are specific for lamins A and C and lamin B, respectively. Lamins A, B, and C are peripheral membrane proteins of the nuclear envelope with structural similarities to cytoplasmic intermediate filament proteins. When studied by indirect immunofluorescence on rat tissues, the serum containing anti-lamin B IgM stained smooth and striated muscles in addition to nuclear envelopes. Lamin B antibodies affinity purified from this serum were able to label muscle cells, suggesting that lamin B shares an epitope(s) with an unidentified muscular component(s). Since in an enzyme-linked immunosorbent assay there was no reactivity with a panel of proteins which are frequent targets of "natural" antibodies, these monoclonal IgM appear to belong to the rare category of IgM that possess a restricted specificity.     

Finding new human minisatellite sequences in the vicinity of long CA-rich sequences.

Microsatellites and minisatellites are two classes of tandem repeat sequences differing in their size, mutation processes, and chromosomal distribution. The boundary between the two classes is not defined. We have developed a convenient, hybridization-based human library screening procedure able to detect long CA-rich sequences. Analysis of cosmid clones derived from a chromosome 1 library show that cross-hybridizing sequences tested are imperfect CA-rich sequences, some of them showing a minisatellite organization. All but one of the 13 positive chromosome 1 clones studied are localized in chromosomal bands to which minisatellites have previously been assigned, such as the 1pter cluster. To test the applicability of the procedure to minisatellite detection on a larger scale, we then used a large-insert whole-genome PAC library. Altogether, 22 new minisatellites have been identified in positive PAC and cosmid clones and 20 of them are telomeric. Among the 42 positive PAC clones localized within the human genome by FISH and/or linkage analysis, 25 (60%) are assigned to a terminal band of the karyotype, 4 (9%) are juxtacentromeric, and 13 (31%) are interstitial. The localization of at least two of the interstitial PAC clones corresponds to previously characterized minisatellite-containing regions and/or ancestrally telomeric bands, in agreement with this minisatellite-like distribution. The data obtained are in close agreement with the parallel investigation of human genome sequence data and suggest that long human (CA)s are imperfect CA repeats belonging to the minisatellite class of sequences. This approach provides a new tool to efficiently target genomic clones originating from subtelomeric domains, from which minisatellite sequences can readily be obtained. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ000377-AJ000383.]     

Identification of the nuclear localization signal of human immunodeficiency virus type 2 Vpx

The Vpx protein of human immunodeficiency virus type 2 (HIV-2) is a viral accessory protein related to, but distinct from, the Vpr protein of HIV-1. Vpx is packaged into virions and, as a component of the viral preintegration complex (PIC), Vpx is required for efficient virus replication in nondividing cells. Therefore, the localization of Vpx in cells is dynamic and dependent upon discrete domains of the protein. Expressed in the absence of other viral proteins, Vpx localizes to the nucleus of cells. However, if expressed with the Gag protein of HIV-2, Vpx localizes to the plasma membrane of cells. To further understand the regulation of Vpx localization, we fused regions of Vpx to beta-galactosidase to identify regions of the protein sufficient to mediate nuclear localization. The minimal transferable region of Vpx that conferred nuclear localization in these assays was aa 65 to 72. Alanine substitution of K(68) and R(70) in a GFP-Vpx construct abolished nuclear localization, suggesting that the basic residues in this region are important for nuclear import. Analysis of the membrane transport of several GFP-Vpx alanine mutants demonstrated that while separable, the domains of Vpx required for nuclear localization are not distinct from the domains required for membrane transport. The results of heterokaryon shuttling assays indicated that Vpx is not a shuttling protein; however, HIV-2 Vpr did shuttle similar to HIV-1 Vpr.     

Inactivators of fibroblast interferon found in human serum.

Investigation of the inactivators of fibroblast interferon has shown that those substances could be identified in every specimen of serum taken from normal individuals. These inactivators were found to destroy a mean of 82% of the interferon antiviral activity beyond that caused by the effects of temperature alone. The inactivators exerted no effect at 4 degrees C but were found in a variety of animal as well as human sera. Although slight differences were found to exist in the extent of inactivation when comparing normal human serum to serum from patients (i) receiving chronic hemodialysis, (ii) with jaundice, or (iii) with underlying malignancies, the differences did not appear to be clinically significant. In exploring the nature of the substances, we found that both dialyzable and non-dialyzable inactivators exist, but the former appear to be the major factors responsible for the unwanted process.     

Complete nucleotide sequences of two RNA segments of human picobirnavirus

Picobirnaviruses are unclassified, non-enveloped, spherical, small viruses with a genome comprising two double-stranded RNA segments. Only incomplete sequence data on picobirnaviruses are available so far. By cloning involving single primer amplification, full-length cDNAs were prepared corresponding to RNA segments 1 and 2 of a picobirnavirus (strain Hy005102) isolated from a stool specimen from an infant with acute non-bacterial gastroenteritis in Thailand, and the complete nucleotide sequences were determined. RNA segments 1 and 2 are 2,525 and 1,745 base pairs in length, respectively. RNA segment 1 encodes two open reading frames (ORFs) of 224 and 552 amino acids, and RNA segment 2 codes for a single ORF of 534 amino acids. On comparison with a part of the nucleotide sequences of the RNA segment, 2 of the other published picobirnavirus strains, the Thai strain was found to be related most closely to one of the US strains.