Hematopoietic cells express two forms of lyn kinase differing by 21 amino acids in the amino terminus

cDNAs for the murine lyn protein tyrosine kinase gene were cloned from mouse bone marrow-derived monocytic cells. Comparison of the human and murine genes demonstrated a 94% homology in peptide sequence. Comparable to the human gene, murine lyn was found to be expressed in myeloid and B-lymphoid lineage cells. During the cloning, two types of cDNAs were obtained that differed by the presence (lynA) or absence (lynB) of 63 bp within the amino-terminal coding region of the gene. The genomic structure of the murine lyn gene demonstrates that the two types of lyn transcripts are derived from alternative splicing utilizing an internal splice donor site. Transcripts for both forms were found to be expressed in myeloid cells. lyn-specific antisera detected comparable levels of proteins of 56 and 53 kDa in hematopoietic cells. these 56- and 53-kDa proteins comigrated with proteins produced by in vitro translation or in vivo expression of the lynA and lynB cDNAs, respectively. The two forms had comparable in vitro kinase activities in immunoprecipitates and showed similar peptide patterns, with partial V8 digestion of the in vitro-phosphorylated proteins. The potential significance of the two lyn proteins is discussed.

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Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3491 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3491-3498 ◽

Cited By ~ 29

Author(s):

R Akada ◽

K Minomi ◽

J Kai ◽

I Yamashita ◽

T Miyakawa ◽

...

Keyword(s):

Tandem Repeats ◽

Rhodosporidium Toruloides ◽

Basidiomycetous Yeast ◽

Peptide Sequence ◽

In Vitro Translation ◽

Mating Pheromone ◽

S1 Nuclease ◽

Amino Terminal ◽

Carboxy Terminal

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.

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Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3491-3498.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3491-3498

Author(s):

R Akada ◽

K Minomi ◽

J Kai ◽

I Yamashita ◽

T Miyakawa ◽

...

Keyword(s):

Tandem Repeats ◽

Rhodosporidium Toruloides ◽

Basidiomycetous Yeast ◽

Peptide Sequence ◽

In Vitro Translation ◽

Mating Pheromone ◽

S1 Nuclease ◽

Amino Terminal ◽

Carboxy Terminal

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.

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Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Genome Medicine ◽

10.1186/s13073-021-00850-w ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

William L. Macken ◽

Annie Godwin ◽

Gabrielle Wheway ◽

Karen Stals ◽

Liliya Nazlamova ◽

...

Keyword(s):

Genome Sequencing ◽

Donor Site ◽

Xenopus Tropicalis ◽

Splice Donor Site ◽

Homologous Region ◽

Disease Genes ◽

Coat Proteins ◽

Splice Donor ◽

Severe Intellectual Disability

Abstract Background Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”. Methods Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2’s mutation. Results We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β’-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi. Conclusions This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

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Identification of protein products encoded by the proto-oncogene int-1.

Molecular and Cellular Biology ◽

10.1128/mcb.7.11.3971 ◽

1987 ◽

Vol 7 (11) ◽

pp. 3971-3977 ◽

Cited By ~ 44

Author(s):

A M Brown ◽

J Papkoff ◽

Y K Fung ◽

G M Shackleford ◽

H E Varmus

Keyword(s):

Retroviral Vectors ◽

Mammary Epithelial ◽

In Vitro Translation ◽

Amino Terminal ◽

Induced Tumors ◽

Epithelial Cell Lines ◽

Tumor Virus ◽

Transforming Activity ◽

The Central Nervous System

The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of all but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.

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Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.99.3.1076 ◽

1984 ◽

Vol 99 (3) ◽

pp. 1076-1082 ◽

Cited By ~ 43

Author(s):

M G Rosenfeld ◽

E E Marcantonio ◽

J Hakimi ◽

V M Ort ◽

P H Atkinson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Posttranslational Modifications ◽

Rat Hepatocytes ◽

Direct Analysis ◽

In Vitro Translation ◽

Endoplasmic Reticulum Membrane ◽

Pituitary Cells ◽

Amino Terminal

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

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Ultrastructural localization of giardins to the edges of disk microribbons of Giarida lamblia and the nucleotide and deduced protein sequence of alpha giardin.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2323 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2323-2335 ◽

Cited By ~ 88

Author(s):

D A Peattie ◽

R A Alonso ◽

A Hein ◽

J P Caulfield

Keyword(s):

Protein Sequence ◽

Protein Identification ◽

Stop Codon ◽

In Vitro Translation ◽

Two Dimensional ◽

One Dimensional ◽

Amino Terminal ◽

Ventral Disk ◽

Dimensional Electrophoresis

The giardins are a group of 29-38-kD proteins in the ventral disk of the protozoan parasite Giardia lamblia. The disk attaches the parasite to the host's intestinal epithelium and is composed of parallel, coiled microtubules that are adjacent to the ventral plasma membrane and from which processes called microribbons extend into the cytoplasm; the microribbons are connected by crossbridges. G. lamblia cytoskeletons, consisting of disks and attached flagella, were isolated and used to show that the 29-38-kD proteins separate into five bands by one-dimensional electrophoresis and into 23 species by two-dimensional analysis. Rabbit antibodies raised against a 33-kD protein band, purified by one-dimensional gel electrophoresis and shown to contain three proteins by two-dimensional electrophoresis, recognized 17 proteins by two-dimensional immunoblot analysis. By immunofluorescence these antibodies reacted with the ventral disk but not with the flagella in isolated cytoskeletons. Electron microscopy revealed that the anti-giardin antibodies bound to the edges of the microribbons but not to the microtubules, crossbridges, or other, nondisk structures. Antibodies to tubulin reacted with both the disk and flagella in isolated cytoskeletons but bound only to the microtubules in these structures. The amino-terminal sequence of the 33-kD immunogen was determined and used to construct a DNA oligomer, and the oligomer was used to isolate the alpha giardin gene. The gene was used to hybrid select RNA, and the in vitro translation product from this RNA was precipitated by the antibodies against the 33-kD immunogen. The gene sequence was a single open reading frame of 885 nucleotides that predicted a protein of 33.8 kD. The protein sequence is unique, having no significant homology to two other giardin sequences or to any sequences within the Protein Identification Resource. It is predicted to be 82% alpha helical. The downstream sequence of the gene indicates that the sequence AGT-PuAA is located six to nine nucleotides beyond the stop codon in all protein-encoding genes of G. lamblia that have been sequenced and reported to date.

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Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains

10.1055/s-0038-1652739 ◽

1981 ◽

Author(s):

G M Fuller ◽

J M Nickerson

Keyword(s):

Signal Peptide ◽

Hepatoma Cell ◽

Temporal Analysis ◽

In Vitro Translation ◽

Translation System ◽

Hepatoma Cell Line ◽

Amino Terminal ◽

Cellular Processes ◽

Microsomal Membranes

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.These studies have been supported in part by NIH HL - 16445 and HL 00162.

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Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.3.c825 ◽

1996 ◽

Vol 271 (3) ◽

pp. C825-C832 ◽

Cited By ~ 16

Author(s):

T. A. Pressley ◽

J. C. Allen ◽

C. H. Clarke ◽

T. Odebunmi ◽

S. C. Higham

Keyword(s):

Insect Cells ◽

In Vitro Translation ◽

Expression Systems ◽

Amino Terminal ◽

Cleavage Process ◽

Heterologous Expression Systems ◽

Terminal Structure ◽

A Site

The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.

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Ribosomal protein L7a is encoded by a gene (Surf-3) within the tightly clustered mouse surfeit locus.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.224 ◽

1989 ◽

Vol 9 (1) ◽

pp. 224-231 ◽

Cited By ~ 29

Author(s):

A Giallongo ◽

J Yon ◽

M Fried

Keyword(s):

Amino Acids ◽

Ribosomal Protein ◽

Ribosomal Subunit ◽

Ribosomal Protein Gene ◽

Mouse Cell ◽

Nucleic Acid Hybridization ◽

Peptide Sequence ◽

In Vitro Translation ◽

Cell Components

The mouse Surfeit locus, which contains a cluster of at least four genes (Surf-1 to Surf-4), is unusual in that adjacent genes are separated by no more than 73 base pairs (bp). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by only 15 to 73 bp, the 3' ends of Surf-1 and Surf-3 are only 70 bp apart, and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp. This very tight clustering suggests a cis interaction between adjacent Surfeit genes. The Surf-3 gene (which could code for a basic polypeptide of 266 amino acids) is a highly expressed member of a pseudogene-containing multigene family. By use of an anti-peptide serum (against the C-terminal nine amino acids of the putative Surf-3 protein) for immunofluorescence and immunoblotting of mouse cell components and by in vitro translation of Surf-3 cDNA hybrid-selected mRNA, the Surf-3 gene product was identified as a 32-kilodalton ribosomal protein located in the 60S ribosomal subunit. From its subunit location, gel migration, and homology with a limited rat ribosomal peptide sequence, the Surf-3 gene was shown to encode the mouse L7a ribosomal protein. The Surf-3 gene is highly conserved through evolution and was detected by nucleic acid hybridization as existing in multiple copies (multigene families) in other mammals and as one or a few copies in birds, Xenopus, Drosophila, and Schizosaccharomyces pombe. The Surf-3 C-terminal anti-peptide serum detects a 32-kilodalton protein in other mammals, birds, and Xenopus but not in Drosophila and S. pombe. The possible effect of interaction of the Surf-3 ribosomal protein gene with adjacent genes in the Surfeit locus at the transcriptional or posttranscriptional level or both levels is discussed.

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