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At least 163 records · Page 9Linked to original sources
PubMed1992

An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (atp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vitro system devised to follow the editing of wheat mitochondrial atp9 mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in vitro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.

Amino Acid Sequence↗
PubMed1991

Cowpea mosaic virus middle component RNA contains a sequence that allows internal binding of ribosomes and that requires eukaryotic initiation factor 4F for optimal translation.

Cowpea mosaic virus (CPMV) middle component RNA (M-RNA) encodes two proteins of 105 and 95 kDa, of which translation starts at nucleotide (nt) 161 and nt 512, respectively. In vitro translation of both proteins directed by T7 transcripts of M-RNA was stimulated fourfold by eukaryotic initiation factor 4F (eIF-4F), the cap-binding protein complex. The ratio of the synthesis of both proteins after translation was not influenced by eIF-4F or by any known eIF. Part of the CPMV 5' sequence was cloned downstream of the 5' untranslated region of ornithine decarboxylase (ODC); the latter untranslated sequence has a highly stable secondary structure, preventing efficient translation of ODC. Insertion of nt 161 to 512 of CPMV M-RNA upstream of the ODC initiation codon resulted in a marked increase in ODC translation, which indicates that the CPMV sequence contains an internal ribosome-binding site. The insertion conferred stimulation by eIF-4F on ODC translation, showing that eIF-4F is able to stimulate internal initiation.

Base Sequence↗
PubMed1992

Genetic and biochemical characterization of the trpB8 mutation of Escherichia coli tryptophan synthase. An amino acid switch at the sharp turn of the trypsin-sensitive "hinge" region diminishes substrate binding and alters solubility.

The trpB8 mutation of Escherichia coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation. The lesion was shown by DNA sequencing to be a G to C transversion at nucleotide 5528 of the trp operon, resulting in a Gly to Arg switch at codon 281. Gly-281, within the trypsin-sensitive "hinge" region, is invariant among all known beta polypeptides. The catalytic activity of the mutant beta 2(B8) protein is dramatically stimulated by alpha subunit, both in vivo and in vitro. In the absence of alpha subunit, ammonium ion effectively stimulated the activity in an apparently cooperative manner. The pH optimum for the mutant subunit was 9.8, which is 2 units higher than that of wild type. In contrast to the wild-type subunit, beta(B8) partially aggregated within cells upon overexpression. At the optimal concentration of ammonium ions (2.25 M), the beta 2(B8) mutant enzyme displayed lower affinity than wild-type enzyme toward indole and L-serine, but the Vmax was almost unchanged. The physicochemical behavior of beta 2(B8) is supported by computer graphic modeling studies. An open versus closed model of conformational change within the beta 2 protein is proposed. A plausible role for the hinge region is discussed.

Amino Acid Sequence↗
PubMed2026

Optimized TadA-derived base editors efficiently manipulate mRNA splicing by A-to-G and C-to-K editing in potato.

Pre-messenger RNA (pre-mRNA) splicing is a critical mechanism for post-transcriptional regulation in plants. Through alternative splicing, plants produce diverse transcriptomes and proteomes that finely regulate development as well as responses to biotic and abiotic stresses. However, modulating the generation of specific splicing isoforms for functional characterization remains challenging, particularly in the non-model crop potato. Here, we show that two optimized TadA-derived base editors efficiently induce diverse mRNA splice variants by targeting specific splice sites. By evaluating multiple adenosine deaminases and performing multi-dimensional optimization, we developed an efficient adenine base editor RTF-ABE8e for potato. RTF-ABE8e achieved 100% editing efficiency at two StDL1 target sites in stable transgenic potato, with homozygous editing frequencies as high as 93.3% and 91.1%, respectively. We also developed RTF-TadDE, a dual-base editor based on a TadA-derived dual deaminase, for A-to-G and C-to-K (K = T/G) mutations in potato with an overall editing efficiency comparable to that of RTF-ABE8e. By targeting different splice sites with these base editors, we obtained diverse splicing isoforms carrying premature termination codons (PTCs) at StDL1 and StPDS and robust mutant phenotypes. These base editors enable efficient and precise editing of splice sites to trigger missplicing, making them powerful tools for manipulating splicing in plants.

Solanum tuberosum↗
PubMed1987

Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a computer program which determined optimal alignment. An amino acid identity of approximately 20% was found between infectious hematopoietic necrosis virus and the two vesicular stomatitis virus serotypes and between infectious hematopoietic necrosis virus and rabies virus. The positions and sizes of the signal sequence and transmembrane domain and the possible glycosylation sites were determined.

Amino Acid Sequence↗
PubMed1992

A point mutation (Ala229 to Thr) in the hinge domain of the c-erbA beta thyroid hormone receptor gene in a family with generalized thyroid hormone resistance.

Various point mutations in the c-erbA thyroid hormone receptor (TR) beta gene of unrelated kindreds have been reported to be responsible for different phenotypes of generalized thyroid hormone resistance. We now report a new point mutation, Td, in one of two TR beta alleles of three affected members of one family, designated family T. In contrast to the previously described point mutations, all located in the T3-binding domain of the TR beta gene, mutation Td was identified in the carboxy-terminal part of the hinge domain. Direct sequencing of the polymerase chain reaction-amplified whole coding region of the patients' fibroblast TR beta genes displayed a single guanine to adenine transition at cDNA nucleotide position 985. This altered alanine (GCC) to threonine (ACC) in codon 229. Garnier prediction of the consequence of the mutation indicated an altered secondary structure. The G----A nucleotide substitution was not present in 80 random TR beta alleles, suggesting that this point mutation is responsible for generalized thyroid hormone resistance in family T. The in vitro expressed mutant TR beta was shown to bind with high affinity to various thyroid hormone response elements. However, the affinity of the TR beta to bind to T3 was reduced 3-fold, indicating that the hinge domain of the TR beta is important for full ligand-binding activity. Moreover, it seems that multiple subdomains of the TR beta interact cooperatively to achieve optimal T3 activity.

Alleles↗
PubMed1987

Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid. We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose. The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis. The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10. The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively. The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors. The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment. The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity. The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene. Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence. Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.

Amino Acid Sequence↗
PubMed1992

An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

Synthesis of the pol gene products of most retroviruses requires ribosomes to shift frame once or twice in the -1 direction while translating gag-pol mRNA. The viral signals for frameshifting include a heptanucleotide sequence on which the shift occurs and higher-order RNA structure just downstream of the shift site. We have made site-directed mutations in two stems (S1 and S2) of a putative RNA pseudoknot that begins 7 nucleotides 3' of the previously identified shift site (A AAA AAC) in the gag-pro region of mouse mammary tumor virus (MMTV) RNA. The mutants confirm the predicted structure, show that loss of either S1 or S2 impairs frameshifting, and exclude alternative RNA structures as significant for frameshifting. The importance of the MMTV pseudoknot has been further demonstrated by showing that shift sites from two other retroviruses function more efficiently in the position of the MMTV site than in their native contexts. However, the MMTV pseudoknot cannot promote detectable frameshifting in the absence of a recognizable upstream shift site. In addition, the species of tRNA that reads the second codon in the shift site appears to be a critical determinant, since changing the 7th nucleotide in the MMTV gag-pro shift site from C to A, U, or G severely impairs frameshifting.

Base Sequence↗
PubMed1978

On the accessibility and selection of the initiator site of mRNA in protein synthesis.

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the "accessibility" of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.

Base Sequence↗
PubMed1985

High-level expression in Escherichia coli of biologically active bovine growth hormone.

High-level synthesis of bovine growth hormone (bGH) in Escherichia coli was achieved by maximizing gene transcription and optimizing the translational efficiency of bGH mRNA. Nearly all of the recombinant hormone was found in the pellet fraction after bacterial cell lysis. This property allowed the purification of bGH nearly to homogeneity. Protein sequence analysis indicated that greater than 93% of the purified hormone had the amino-terminal methionine residue removed by E. coli, yielding mature bGH. In a hypophysectomized rat assay system, purified bacterial-produced bGH demonstrated growth-promoting activity equivalent to that of pituitary-derived bovine growth hormone.

Amino Acid Sequence↗
PubMed2026

seq2ribo: structure-aware integration of machine learning and simulation to predict ribosome location profiles from RNA sequences.

MOTIVATION: Ribosome dynamics are vital in the process of protein expression. Current methods rely on ribosome profiling (Ribo-seq), RNA-seq profiles, and full genomic context. This restricts their use in de novo sequence design, like messenger RNA (mRNA) vaccines. Simulation-only approaches like the Totally Asymmetric Simple Exclusion Process (TASEP) oversimplify translation by focusing solely on codon elongation times. RESULTS: We present seq2ribo, a hybrid simulation and machine learning framework that predicts ribosome A-site locations using only an mRNA sequence as input. Our method first employs a novel structure-aware TASEP (sTASEP), which models translation using a comprehensive set of fitted parameters that include codon wait times and structural features, such as local angles, base-pairing, and discrete positional buckets. The ribosome locations generated by sTASEP are then processed by a polisher model, which learns to refine the simulated ribosome distributions. seq2ribo provides high-fidelity predictions of ribosome locations across diverse cell types (iPSC, HEK293, LCL, and RPE-1), significantly outperforming baselines. seq2ribo is the first method to achieve meaningful positional correlation with observed ribosome profiles from sequence alone, reaching transcript-level Pearson correlations up to 0.920 and within-transcript shape correlations up to 0.186, where all baselines yield near-zero values on these metrics. seq2ribo also reduces elementwise error by up to 37.7% relative to the sequence-only Translatomer baseline. By adding a task-specific head, seq2ribo achieves Pearson correlations up to 0.732 with experimental translation efficiency (TE) across several cell lines, and up to 0.903 with measured protein expression. By operating from sequence alone, seq2ribo provides a new tool for synthetic biology, enabling the rational design and optimization of mRNA sequences without the need for expression-level data or genomic context. AVAILABILITY: seq2ribo is available at https://github.com/Kingsford-Group/seq2ribo.

Machine Learning↗
PubMed2026

seq2ribo: Structure-aware integration of machine learning and simulation to predict ribosome location profiles from RNA sequences.

MOTIVATION: Ribosome dynamics are vital in the process of protein expression. Current methods rely on ribosome profiling (Ribo-seq), RNA-seq profiles, and full genomic context. This restricts their use in de novo sequence design, like messenger RNA (mRNA) vaccines. Simulation-only approaches like the Totally Asymmetric Simple Exclusion Process (TASEP) oversimplify translation by focusing solely on codon elongation times. RESULTS: We present seq2ribo, a hybrid simulation and machine learning framework that predicts ribosome A-site locations using only an mRNA sequence as input. Our method first employs a novel structure-aware TASEP (sTASEP), which models translation using a comprehensive set of fitted parameters that include codon wait times and structural features, such as local angles, base-pairing, and discrete positional buckets. The ribosome locations generated by sTASEP are then processed by a polisher model, which learns to refine the simulated ribosome distributions. seq2ribo provides high-fidelity predictions of ribosome locations across diverse cell types (iPSC, HEK293, LCL, and RPE-1), significantly outperforming baselines. seq2ribo is the first method to achieve meaningful positional correlation with observed ribosome profiles from sequence alone, reaching transcript-level Pearson correlations up to 0.920 and within-transcript shape correlations up to 0.186, where all baselines yield near-zero values on these metrics. seq2ribo also reduces elementwise error by up to 37.7% relative to the sequence-only Translatomer baseline. By adding a task-specific head, seq2ribo achieves Pearson correlations up to 0.732 with experimental translation efficiency (TE) across several cell lines, and up to 0.903 with measured protein expression. By operating from sequence alone, seq2ribo provides a new tool for synthetic biology, enabling the rational design and optimization of mRNA sequences without the need for expression-level data or genomic context.

Journal Article↗
PubMed1987

The relationship between the biosynthetic paths to the amino acids and their coding. I: The aliphatic amino acids and proline.

The genetic code could not have been fixed until the means for biosynthesis of the amino acids was at hand. The biosynthetic enzymes could not be optimized until the genetic code ceased to be rearranged. Therefore the development of the code and the development of the biosynthesis of the amino acids occurred concurrently. The present day biosynthetic pathways of amino acids, examined from this point of view, help to explain the present set of coded amino acids, in particular the absence of norvaline, norleucine, homoserine, ornithine, and alpha-aminobutyric acid. An order of development of biosyntheses is also proposed. Lysine was first, followed by valine and isoleucine. The more common primordial amino acids did not need biosyntheses so early. The central pathways of metabolism probably developed in response to a need for amino acid biosynthesis.

Amino Acids↗
PubMed1992

Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts.

The envelope of the hepatitis B virus (HBV) is composed of three species of proteins, the large (L), middle (M), and major (S) surface proteins (HBsAgs), each of different molecular weights but sharing a common C-terminus. These three HBsAgs, encoded by two species of viral subgenomic transcripts (2.1 and 2.4 kb), which have a heterogenous 5'-terminus, appear in different amounts in both the 42-nm virions and the 22-nm subviral particles. To investigate the involvement of translational control in the differential expression of the L, M, and S proteins, we tested the translational capability of 2.1- and 2.4-kb transcripts in in vitro translation and of 2.1-kb transcripts in in vivo transfection experiments. Results of in vitro translation indicated that a large amount of the L protein and a very small amount of the M and S proteins were synthesized from the 2.4-kb mRNA. Translation of the 2.1-kb mRNA resulted in a 4:1 ratio of the S protein to the M protein. In contrast, translation of a similar 2.1-kb mRNA containing an optimal initiation context (5'-ACCATGG-3') of the pre-S2 region resulted in a reversed ratio, four times as much M protein as S protein. This result was also obtained by transfection of hepatoma cells with plasmid DNAs containing the mutated sequence (5'-ACCATGG-3') of the pre-S2 region. In considering these results, the production of a large amount of the L protein from the 2.4-kb mRNA and the determination of the level of the M protein by the context of translational initiation, we suggest that a preferential translational initiation is involved in the expression of differential amounts of the L, M, and S proteins.

Base Sequence↗
PubMed1992

Molecular analysis of the Haemophilus ducreyi groE heat shock operon.

Chancroid is a sexually transmitted genital ulcer disease caused by Haemophilus ducreyi. Previously, we developed diagnostic DNA probes for H. ducreyi (L. M. Parsons, M. Shayegani, A. L. Waring, and L. H. Bopp, J. Clin. Microbiol. 27:1441-1445, 1989). In the present study, DNA sequencing of one of the diagnostic probes revealed two adjacent open reading frames (ORFs). These H. ducreyi ORFs and the encoded proteins show significant homology with the groE genes and GroES and GroEL heat shock proteins from several bacterial pathogens and with conserved eukaryotic 60-kDa heat shock proteins. The first H. ducreyi ORF (groES) is preceded by sequences similar to those of the Escherichia coli consensus heat shock promoters and is 288 nucleotides long and is capable of encoding a protein of 10.3 kDa. The second ORF (groEL) is 1,641 nucleotides long and is capable of encoding a protein of 57.8 kDa. Northern (RNA blot) analysis demonstrated the presence of a high level of groE mRNA in exponential-phase H. ducreyi grown in hemin broth at the organism's optimal growth temperature (33 degrees C), with increased levels seen following heat shock. Heat shock also increased the thermostability of the organisms, since stressed cells were more resistant to the lethal effects of rapid chilling. Electrophoretic analysis and immunoblots demonstrated that the predominant protein produced by exponential-phase H. ducreyi was a heat-inducible, immunoreactive protein of approximately 60 kDa (GroEL). Also, H. ducreyi groE mRNA and GroEL were expressed and inducible by heat in E. coli. This is the first report describing the cloning, sequencing, and expression of H. ducreyi protein-encoding genes.

Amino Acid Sequence↗
PubMed1992

Memory T cells of a patient with follicular thyroid carcinoma recognize peptides derived from mutated p21 ras (Gln-->Leu61).

Point mutations in ras genes resulting in substitutions of amino acid Gly in positions 12 and 13, and Gln in position 61 of the ras gene product p21, are commonly found in human tumors. Peptides derived from aberrant p21 may elicit a tumor specific T cell response, provided that these peptides can bind to HLA molecules of the tumor and the patient has T cells able to recognize the corresponding peptide-HLA complex. Here we report that CD4+ T cells of memory type (CD45RO+) from a patient with a follicular thyroid carcinoma respond against a synthetic peptide derived from aberrant p21 ras having a Gln-->Leu substitution at position 61. Such responses were not observed when T cells from healthy volunteers or cancer patients where this mutation does not usually occur were stimulated with this peptide. The responding T cells did not cross-react with the corresponding peptide derived from native p21 ras nor did they recognize peptides carrying other substitutions in position 61. T cells clones were generated which recognized this Leu61 peptide when presented by HLA-DQ8 molecules. These T cell clones also recognized the corresponding intact p21 ras protein. By using several different synthetic peptides, a peptide with optimal stimulatory capacity was defined. Performing polymerase chain reaction and oligonucleotide probing we were, however, not able to detect the p21 ras gene encoding the Gln-->Leu substitution in DNA from tumor biopsies from the patient. This may indicate that tumor cells harboring the mutation leading to the Gln-->Leu substitution had been eliminated and that tumor progression was due to cells that had deleted the mutated ras gene. The finding that ras derived peptides and recombinant mutated p21 ras are immunogenic in man may form the basis for the development of cancer immunotherapy based on synthetic oncogene derived peptides.

Adenocarcinoma↗
PubMed1976

The involvement of the anticodon adjacent modified nucleoside N-(9-(BETA-D-ribofuranosyl) purine-6-ylcarbamoyl)-threonine in the biological function of E. coli tRNAile.

tRNAile was isolated from E. coli Cp 79 (leu-, arg-, thr-, his-, thiamin-, RCrel) which had been grown on a sub-optimal concentration of thr and was found to contain an average of 50% less N-[9-(beta-D-ribofuranosyl)- purin-6-ylcarbamoyl]threonine, t6Ado, than tRNAile from cells grown on an optimum concentration of thr and containing a normal complement of t6Ado. The two tRNA's were identical in their ability to be aminoacylated, to accept the 3'-terminal dinucleotide, and to form an ile-tRNAile-Tu-GTP complex. In contrast, the t6Ado-deficient-tRNA was significantly less efficient in binding to ribosomes compared to the normal tRNA. This difference was seen in the binding of deacylated tRNA and in the nonenzymatic and enzymatic binding of ile-tRNA, all in response to poly AUC. The t6Ado-deficient ile-tRNA demonstrated no binding at Mg2+ concentrations less than or equal to 10 mM, while the normal ile-tRNA bound at low Mg2+ concentrations. Tetracycline had the same effect on the normal as on the t6Ado-deficient ile-tRNA binding. As a control, the binding of phe-tRNA (which does not contain t6Ado) from normal and thr-starved cells in response to poly U was identical. It was concluded that t6Ado is required for proper codon-anticodon interaction.

Amino Acyl-tRNA Synthetases↗
PubMed1991

Analysis of Fc gamma RIII (CD16) membrane expression and association with CD3 zeta and Fc epsilon RI-gamma by site-directed mutation.

Two genes encode Fc gamma RIII (CD16), a low affinity FcR for IgG. CD16-I is expressed as a phosphatidylinositol glycan-anchored membrane glycoprotein on neutrophils, whereas CD16-II is a transmembrane-linked glycoprotein on NK cells. Membrane anchoring is determined by codon 203. Site-directed mutation of codon 203 and transient expression of these cDNA in COS-7 cells indicated that Phe, Ile, Leu, and Val permit transmembrane expression, whereas Ser, Thr, Tyr, Asn, Gly, Ala, Asp and Lys enable phosphatidylinositol-glycan attachment. Thus, the involvement of amino acid 203 in membrane anchoring cannot be explained simply on the basis of size, charge, or polarity of the amino acid side groups at this site. Efficient expression of CD16-II in COS-7 cells requires co-transfection with either CD3 zeta or Fc epsilon RI-gamma. Truncation of the cytoplasmic segment of CD16 failed to affect association with CD3 zeta. CD3 zeta and Fc epsilon RI-gamma with truncated cytoplasmic segments were also able to facilitate membrane expression of CD16-II, implicating the transmembrane segments as the interaction site between CD16-II and CD3 zeta or Fc epsilon RI-gamma. Prior studies have suggested that the acidic residue in the CD3 zeta transmembrane segment may be important for the association of CD3 zeta complexes. Although site-directed mutation of CD3 zeta-Asp36 to Glu, Leu, or Val retained the ability to permit membrane expression of CD16-II, quantitatively the wild-type CD3 zeta-Asp36 provided optimal levels of expression, consistent with conservation of this amino acid in mouse and human CD3 zeta.

Antigens, CD↗