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P D Baas

Publications and source records attributed to P D Baas.

At least 19 recordsLinked to original sources
PubMed1998

Expression of the receptor tyrosine kinase Ret on the plasma membrane is dependent on calcium.

Mutations in the Ret receptor tyrosine kinase are responsible for a variety of human syndromes, including multiple endocrine neoplasia 2 and Hirschsprung's disease. Ret is expressed as a 150-kDa precursor form in the endoplasmic reticulum and a 170-kDa mature form at the plasma membrane. Here we show that expression of p170(ret) is dependent on calcium. Depletion of extracellular calcium completely blocks p170(ret) expression, which is not caused by a decrease in half-life of p170(ret) at the plasma membrane but by a defect in processing of p150(ret) into p170(ret). This processing defect can be mimicked by treating the cells with thapsigargin, a drug that releases calcium from internal stores, indicating that reduction in luminal calcium is responsible for the processing defect. We propose that a relatively high concentration of luminal calcium is necessary for the proper folding of Ret in the endoplasmic reticulum.

Calcium↗
PubMed1995

Ret receptor tyrosine kinase activates extracellular signal-regulated kinase 2 in SK-N-MC cells.

Ret is a receptor tyrosine kinase predominantly expressed in tissue derived from the neuroectoderm and is involved in multiple endocrine neoplasia type 2A and 2B, familiar medullary thyroid carcinoma, and Hirschsprung's disease. The ligand for the receptor is still unknown. Previously, using a human epidermal growth factor receptor - Ret chimaeric receptor (HERRet) stably transfected into fibroblasts, it was shown that Ret activation induces the activation of p21ras, but, surprisingly, activation of extracellular signal-regulated kinase 2 (ERK2) was not observed (Santoro et al. (1994) Mol. Cell. Biol., 14, 663). In this report we describe early signaling events induced by the activated HERRet fusion receptor in a cell line derived from neuroectodermal tissue, SK-N-MC. In these cells, activated HERRet induces tyrosine phosphorylation of Shc, complex formation of Shc with Grb2 and Sos and activation of p21ras. Importantly, also ERK2 is activated. This activation was strong and sustained for at least 2 h. Activation was abolished by the dominant negative p21rasasn17 mutant, showing that activation of ERK2 is mediated by p21ras. These results suggest that Ret can induce ERK2 activation in a p21ras dependent manner in cells derived from tissue where Ret is endogenously expressed.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Cooperation of 5' and 3' processing sites as well as intron and exon sequences in calcitonin exon recognition.

We have previously shown that the calcitonin (CT)-encoding exon 4 of the human calcitonin/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is surrounded by suboptimal processing sites. At the 5' end of exon 4 a weak 3' splice site is present because of an unusual branch acceptor nucleotide (U) and a weak poly(A) site is present at the 3' end of exon 4. For CT-specific RNA processing two different exon enhancer elements, A and B, located within exon 4 are required. In this study we have investigated the cooperation of these elements in CT exon recognition and inclusion by transient transfection into 293 cells of CALC-I minigene constructs. Improvement of the strength of the 3' splice site in front of exon 4 by the branchpoint mutation U-->A reduces the requirement for the presence of exon enhancer elements within exon 4 for CT-specific RNA processing, irrespective of the length of exon 4. Replacement of the exon 4 poly(A) site with a 5' splice site does not result in CT exon recognition, unless also one or more exon enhancer elements and/or the branchpoint mutation U-->A in front of exon 4 are present. This indicates that terminal and internal exons are recognised in a similar fashion. The number of additional enhancing elements that are required for CT exon recognition depends on the strength of the 5' splice site. Deletion of a large part of intron 4 also leads to partial exon 4 skipping. All these different elements contribute to CT exon recognition and inclusion. The CT exon is recognised as a whole entity and the sum of the strengths of the different elements determines recognition as an exon. Curiously, in one of our constructs a 5' splice site at the end of exon 4 is either ignored by the splicing machinery of the cell or recognised as a splice donor or as a splice acceptor site.

Animals↗
PubMed1994

The exon 4 poly(A) site of the human calcitonin/CGRP-I pre-mRNA is a weak site in vitro.

The human calcitonin/CGRP-I (CALC-I) pre-mRNA is processed in a tissue-specific alternative way into either calcitonin (CT) or calcitonin gene-related peptide-I (CGRP-I) mRNA. The exons 1 to 3 are common exons. They are spliced to exon 4, which becomes polyadenylated to form CT mRNA, or to exon 5 and the polyadenylated exon 6 to form CGRP-I mRNA. Polyadenylation at exon 4 and splicing of exon 3 to exon 5 are mutually exclusive processing reactions. Only splicing of exon 3 to exon 5 was detected in vitro, with a minigene containing the exon 3 to exon 5 region. No polyadenylation at the exon 4 poly(A) site could be observed. Investigation of the properties of the exon 4 poly(A) site in vitro shows that it is inefficiently used in vitro. Cleavage and polyadenylation of short RNAs containing only the exon 4 poly(A) site is strongly dependent on the 3' length of the RNA. Downstream sequences located within 39 nucleotides from the cleavage site are required for optimal cleavage and polyadenylation. When the exon 4 poly(A) site in the minigene is replaced with the strong adenovirus L3 or rabbit beta-globin poly(A) sites, these sites can be efficiently used in vitro.

Alternative Splicing↗
PubMed1994

Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA.

The calcitonin (CT)/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is subject to alternative tissue-specific processing of its primary transcript. CT mRNA is the predominant mRNA produced in thyroid C cells, whereas CT gene-related peptide I mRNA is the main product in neurons of the central and peripheral nervous systems. The CT-specific exon 4 is surrounded by weak processing sites. In this study we have investigated whether exon 4 sequences are involved in the tissue-specific selection of the exon 4 splice acceptor site. The results indicate that two separate elements, termed A and B, in the 5' part of exon 4 are required for production of CT-specific RNA. These sequences are located between nucleotides 67 and 88 (A) and nucleotides 117 and 146 (B) relative to the 5' end of exon 4. Variation of the distance between these sequence elements and the 3' splice site of exon 4 does not change the processing choice. These sequence elements are functionally equivalent. CT-specific splicing requires the presence of both sequence A and B or duplicates of either sequence element in exon 4. The effect of these sequences on the RNA processing choice is overruled by mutation of the CT-specific uridine branch acceptor nucleotide into a commonly preferred adenosine residue.

Alternative Splicing↗
PubMed1993

A PCR-based method for the analysis of human CD44 splice products.

CD44 is a transmembrane glycoprotein involved in the interaction between cells and extracellular matrix. Several variant forms of CD44 exist, which differ from each other in the composition of both the intra- and extracellular domain of the protein. Post-translational modification and alternative RNA processing are responsible for this variation. Recently, it was found that certain variant CD44 proteins, containing extra sequences in the extracellular domain of the protein, are involved in metastatic spread of tumor cells. Variant CD44 proteins are also involved in immunological functions of T and B cells. A large variety of alternatively spliced CD44 mRNAs can be expressed by cells. We have developed a method for the analysis of CD44 mRNAs present in the cell. This reverse transcription-polymerase chain reaction (RT-PCR)-based method can be used to analyze the exon composition of each major CD44 mRNA species present in the cell. In this study we describe the analysis of CD44 mRNAs isolated from six different human cell lines.

Antigens, CD↗
PubMed1992

A novel calcitonin-encoding mRNA is produced by alternative processing of calcitonin/calcitonin gene-related peptide-I pre-mRNA.

The calcitonin/calcitonin gene-related peptide-I (CALC-I) gene was one of the first examples of a cellular gene exhibiting alternative, tissue-specific processing of its primary RNA transcript. Calcitonin (CT) mRNA is the predominant product in thyroid C cells, whereas calcitonin gene-related peptide-I mRNA is the main product in neurons of the central and peripheral nervous systems. In this study, we report the existence of a novel CT-like (CT*) mRNA derived from the human CALC-I gene. CT* mRNA is the result of a novel splicing reaction which was initially detected in vitro. Translation of CT* mRNA will result in a prohormone (proCT*) which only differs from proCalcitonin (proCT) in its carboxyl terminus. Proteolytic processing of proCT* predicts, beside the amino-terminal cleavage peptide and CT, the existence of a novel carboxyl-terminal protein of 21 amino acids.

Amino Acid Sequence↗
PubMed1991

Deregulation of alternative processing of Calcitonin/CGRP-I pre-mRNA by a single point mutation.

The Calcitonin/CGRP-I (CALC-I) gene was one of the first examples of a cellular gene exhibiting alternative, tissue-specific processing of its primary transcript. Calcitonin (CT) mRNA is the predominant product in thyroid C-cells, whereas CGRP-I (Calcitonin Gene Related Peptide-I) mRNA is the main product in neurons of the central and peripheral nervous systems. Investigating the molecular mechanism underlying the alternative processing events, we have demonstrated that the CT-specific splice acceptor site is an intrinsical weak site due to usage of a uridine branch acceptor. The data presented in this report show that a single point mutation changing the uridine branch acceptor into a commonly preferred adenosine residue results in the predominant production of CT mRNA in otherwise CGRP-I mRNA-producing F9 cells. The results of the experiments implicate that the low efficiency of CT splicing, caused by usage of a uridine branch acceptor, allows the production of CGRP-I mRNA in neural cells.

Animals↗
PubMed1990

Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man.

Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene. Both consist of three exons and two introns which are very similar. The upstream sequence of the rat IAPP gene contains a TATA-box, a CCAAT-sequence and a GT-element, whereas the upstream sequence of the human IAPP gene contains a TATA-box and a rat insulin enhancer-like sequence. This suggests that the rat and human IAPP gene may be controlled differently at the transcriptional level.

Amyloid↗
PubMed1990

Uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of calcitonin/CGRP-l pre-mRNA.

The human calcitonin/CGRP-I (CALC-I) gene contains 6 exons and encodes two polypeptide precursors. In thyroid C-cells, calcitonin (CT) mRNA is produced by splicing of exons 1-2-3 to exon 4 (CT-encoding) and polyadenylation at exon 4. CGRP-I mRNA is produced in particular neural cells by splicing of exons 1-2-3 to exon 5 (CGRP-I-encoding) and the polyadenylated exon 6. We previously reported that model precursor RNAs containing the exon 3 to exon 5 region of the CALC-I gene are processed predominantly into CGRP-I mRNA in vitro, in nuclear extracts of several cell types (neural and non-neural). Using truncated precursor RNAs containing only the exon 3 to exon 4 region of the CALC-I gene it was shown that CT splicing is an inefficient reaction in which a uridine residue serves as the major site of lariat formation. Here we report that the low CT splicing efficiency and the dominance of CGRP-I splicing over CT splicing in vitro are primarily due to the usage of the CT-specific uridine branch acceptor. Mutation of this uridine residue into an adenosine residue resulted in a strong increase in CT splicing efficiency causing a reversal of the splicing pattern. In addition, it was shown that this point mutation also increased CT splicing efficiency in vivo. These results and data obtained from other experiments involving mutation of the CT splice acceptor site suggest that the uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of the CALC-I pre-mRNA.

Adenosine↗
PubMed1989

In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA.

The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene. A precursor RNA containing the full-length exon 3 to exon 5 region was only poorly spliced in vitro. Therefore, a systematic analysis was performed of the effect of deletions introduced in the intron 3, exon 4 and intron 4 of this precursor RNA on calcitonin/CGRP mRNA-specific splicing. The deletions increased the efficiency of splicing considerably. In all cases CGRP mRNA-specific splicing is strongly favoured over calcitonin mRNA-specific splicing. In addition, splicing reactions using cryptic 5' splice sites were detected which interfered with the usage of processing signals for calcitonin and CGRP mRNA-specific splicing. The results imply a major regulatory role for the exon 4 poly(A) addition reaction in the generation of calcitonin mRNA.

Calcitonin↗
PubMed1988

Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA.

To study splice site selection in alternative RNA processing we used the human Calcitonin/CGRP-I (CALC-I) gene. Expression of the CALC-I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (containing exons 1 to 4) whereas CGRP-I mRNA (containing exons 1,2,3,5 and 6) is the exclusive product in particular nerve cells. We previously reported that a model precursor RNA containing the exon 3 to exon 5 region is predominantly processed into CGRP-I mRNA in vitro using nuclear extracts of three different cell types. To study CT specific processing in Hela cell nuclear extracts we have used precursor RNAs corresponding to the exon 3 to exon 4 region containing only CT specific processing signals. The results revealed the usage of a uridine residue 23 nucleotides upstream of the 3' splice site as the major site of lariat formation in CT specific splicing. The implications of this finding for the alternative, tissue specific processing of the CALC-I pre-mRNA and for branch point selection in general are discussed.

Calcitonin↗
PubMed1988

Model for tissue specific Calcitonin/CGRP-I RNA processing from in vitro experiments.

The Calcitonin/CGRP-I (CALC-I) gene is known to be expressed in a tissue specific fashion resulting in the production of Calcitonin mRNA in thyroid C-cells and CGRP-I mRNA in particular nerve cells. The alternative RNA processing reactions include splicing of exons 1, 2 and 3 to exon 4 and poly (A) addition at exon 4 (Calcitonin mRNA) or splicing of exons 1, 2 and 3 to exons 5 and 6 and poly (A) addition at exon 6 (CGRP-I mRNA). Using a model precursor RNA containing the exon 3 to exon 5 region of the human CALC-I gene we have investigated the Calcitonin- and CGRP-I mRNA-specific processing reactions in vitro, in nuclear extracts of Hela, PC12 and Ewing-1B cells, respectively. Extracts of PC12- and Ewing-1B cells were expected to perform CGRP mRNA-specific splicing, whereas Calcitonin mRNA specific processing was expected to occur in Hela cell extracts. Surprisingly, CGRP mRNA-specific splicing of exon 3 to exon 5 was the predominant reaction in all three extracts. Significant Calcitonin mRNA-specific splicing of exon 3 to exon 4 only took place upon elimination of the dominant downstream 3' splice site used in CGRP mRNA-specific splicing. This elimination occurs most definitively by cleavage at the Calcitonin mRNA specific poly (A) site at exon 4 which may then be the major regulatory mechanism for tissue-specific expression of the CALC-I gene.

Calcitonin↗
PubMed1987

Mutational analysis of the bacteriophage phi X174 replication origin.

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.

Bacteriophage phi X 174↗
PubMed1987

Alteration of the ATG start codon of the A protein of bacteriophage phi X174 into an ATT codon yields a viable phage indicating that A protein is not essential for phi X174 reproduction.

Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable phi X174 mutant was constructed in which the ATG start codon of the A protein was changed into an ATT codon. This mutant, phi X-4499T, does not synthesize A protein. The burst size of phi X-4499T amounted to 50% of that of wild type phi X174. This indicates that A protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage phi X174.

Bacteriophage phi X 174↗
PubMed1986

Model for alternative RNA processing in human calcitonin gene expression.

The alternative RNA processing pathways in human calcitonin gene (CALC-I gene) expression were investigated using steady state RNA isolated from human medullary thyroid carcinoma (MTC) and from a culture line derived from this tumor. On Northern blots the mature 1.0 kilobases (Kb) calcitonin (CT) - and 1.1 Kb calcitonin gene-related peptide (CGRP) mRNAs were detected with CALCI gene specific probes as well as high molecular weight poly (A) containing RNAs of 2.1, 2.3, 3.3, 4.2, 5.0 and 5.7 Kb. The 5.7 Kb RNA was identified as the poly(A) tailed primary transcript containing sequences corresponding to all 6 exons and 5 introns of the CALC-I gene. From the composition of the other RNAs the splicing order of the different introns could be deduced. The results suggest the following model. First all introns not involved in alternative processing (introns 1, 2 and 5) are spliced from the 5.7 Kb RNA in rapid successive reactions yielding a 3.3 Kb RNA, which accumulates. From this 3.3 Kb RNA, the last common intermediate in the alternative processing pathway, CT mRNA is formed by splicing of intron 3 and poly(A) addition at exon 4, in this order or the reverse order via 2.3 Kb or 2.1 Kb RNA intermediates respectively. Alternatively, the whole intron 3-exon 4-intron 4 region is spliced from the 3.3 Kb RNA yielding CGRP mRNA. The temporal sequence of poly(A) addition at exons 4 and 6 may relate to the observed structural differences between the poly(A) addition signals at these sites. The ratio of CT- to CGRP mRNA may relate also to the differences in the primary structures of the intron 3- and intron 4 splice acceptor sites.

Base Sequence↗
PubMed1986

Termination and reinitiation signals of bacteriophage phi X174 rolling circle DNA replication.

The nucleotide sequence requirements for termination and reinitiation of rolling circle DNA replication within the 30-bp phi X174 origin region were studied. Plasmids were constructed which contained a complete and a partial phi X174 origin region in the same orientation. The partial origin consisted of the first 16, 24, 25, 26, 27, or 28 bp of the origin region. Plasmids harboring a complete origin region are subject to rolling circle DNA replication and packaging of single-stranded plasmid DNA into phage coats in phi X174 or G4 phage infected cells. The plasmids with a complete and partial origin region were tested in these in vivo transduction systems. The results lead to the following conclusions: The phi X174 and G4 in vivo transduction systems are useful in studying termination and reinitiation of rolling circle DNA replication. The first 24 bp of the origin region are sufficient for termination of a round of rolling circle DNA replication coupled to DNA packaging. The first 16 bp, however, are not recognized as a termination signal. Reinitiation of rolling circle DNA replication coupled to DNA packaging on a partial origin region occurs with low frequency.

Bacteriophage phi X 174↗