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C C Garner

Publications and source records attributed to C C Garner.

13 recordsLinked to original sources
PubMed1992

Microtubule-associated proteins 1A and LC2. Two proteins encoded in one messenger RNA.

The deduced amino acid sequence for the filamentous microtubule-associated protein (MAP) 1A, thought to be involved in stabilizing the mature neuronal cytoskeleton, has been determined from a series of overlapping cDNA clones. Though previously described as biochemically and immunologically distinct from MAP1B, we now demonstrate that MAP1A is structurally related to MAP1B, a protein associated with neurite outgrowth and process plasticity. The two MAPs exhibit regional amino acid sequence similarities spanning their potential microtubule binding domains placing both into a new MAP family. The cDNA sequence encoding MAP1A was also found to encode one of its associated light chains (LC) called LC2. Both proteins are found on a single mRNA in the same open reading frame and are translated as a pre-MAP1A/LC2-protein. The topological relationship between MAP1A and LC2 coding sequences is, therefore, identical to that previously shown for MAP1B and LC1 (Hammarback, J. A., Obar, R. A., Hughes, S. M., and Vallee, R. B. (1991) Neuron 7, 129-139). Based on these and earlier results, we conclude that LC1 and LC2 are structurally related polypeptides generated from distinct MAP polyprotein precursors but free to exchange between the two MAPs.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of microtubule-associated proteins tau and MAP2.

Tau and MAP2 are two of the major microtubule-associated proteins in the vertebrate nervous system. They promote microtubule assembly and stability, and might be involved in the establishment and maintenance of neuronal polarity. In nerve cells immunohistochemistry shows complementary distributions, with tau being concentrated in axons and high molecular mass MAP2 being confined to dendrites. Each protein consists of multiple isoforms that contain three or four homologous tandem repeats near the carboxy-terminus, which constitute microtubule-binding domains. In humans, tau consists of at least six isoforms of related amino acid sequences that are produced from a single gene by alternative mRNA splicing and that are expressed in a stage- and cell type-specific manner. Tau is also a component of the paired helical filaments associated with Alzheimer's disease and other disorders of the CNS. Rat MAP2 consists of at least three isoforms produced from a single gene: high molecular mass MAP2a and MAP2b, and low molecular mass MAP2c. MAP2c is expressed only during early development and has so far been seen only in axons; MAP2a appears to replace MAP2c, whereas MAP2b is expressed throughout life. Messenger RNAs for MAP2 of high molecular mass are expressed both in cell bodies and in dendrites, consistent with the dendritic localization of the corresponding protein isoforms.

Amino Acid Sequence↗
PubMed1990

Molecular structure of microtubule-associated protein 2b and 2c from rat brain.

Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced. The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli. The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain. The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2. This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence. Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively. A third homologous domain of unknown function lies in a central region of MAP2b. Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2. Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of microtubule-associated protein 1 (MAP1A) and microtubule-associated protein 5 (MAP1B): identification of distinct genes and their differential expression in developing brain.

cDNA clones encoding microtubule-associated proteins 1 (MAP1/MAP1A) and 5 (MAP5/MAP1B) were isolated and have been used to study their structural relationship as well as their regulated expression in developing rat brain. cDNA clones specific for MAP1 hybridized to a single 10-kb rat brain mRNA, and analysis of genomic DNA by Southern blotting indicated the existence of a single MAP1 gene. A second set of cDNAs specific for MAP5 hybridized to a single 11-kb mRNA in rat brain and also detected a single gene. By analysis of hybrid mouse-hamster cell lines, the MAP1 gene was located to mouse chromosome 2, designated Mtap-1, and the MAP5 gene to chromosome 13, designated Mtap-5. MAP1 and MAP5 mRNAs were expressed with different temporal patterns during rat brain development that mirrored the appearance of their protein products, suggesting that expression of these proteins is under transcriptional control. These results taken together demonstrate that although MAP1 and MAP5 have some properties that are similar, they are structurally distinct proteins whose transcription is differently regulated from separate genes.

Animals↗
PubMed1989

Embryonic MAP2 lacks the cross-linking sidearm sequences and dendritic targeting signal of adult MAP2.

The most prominent microtubule-associated protein of the neuronal cytoskeleton is MAP2. In the brain it exists as a pair of high-molecular weight proteins, MAP2a and MAP2b, and a smaller form, MAP2c, which is particularly abundant in the developing brain. High-molecular weight MAP2 is expressed in dendrites, where its messenger RNA is also located, but is not found in axons; it has been shown to be present in fine filaments that crosslink dendritic microtubules. This correlates with the primary structure of high-molecular weight MAP2, which consists of a short carboxy-terminal tubulin-binding domain and a long amino-terminal arm, which forms a filamentous sidearm on reconstituted microtubules. Here we report that the high- and low-molecular weight forms of MAP2 are generated by alternative splicing and share the entire C-terminal tubulin-binding domain as well as a short N-terminal sequence. In contrast to high molecular weight MAP2, embryonic brain MAP2c lacks 1,342 amino acids from the filamentous sidearm domain. Furthermore, the mRNA for low molecular weight MAP2c is not present in dendrites, indicating that the dendritic targeting signal is specific for the high-molecular weight form.

Amino Acid Sequence↗
PubMed1989

Microtubule-associated proteins MAP5 and MAP1x: closely related components of the neuronal cytoskeleton with different cytoplasmic distributions in the developing brain.

Monoclonal antibodies were used to explore the relationship between two similarly sized microtubule-associated proteins (MAPs), MAP1x and MAP5. Although the proteins detected by anti-MAP1x and anti-MAP5 co-migrate in SDS-polyacrylamide gels, the patterns of antigenic proteolytic fragments (epitope maps) derived from them were completely different. The results suggest either that MAP1x is more stable than MAP5 or that the MAP1x epitope is situated close to one end of the molecule and gives rise to a very short proteolytic fragment. Immunoprecipitation from brain supernatants with either antibody brought down protein that cross-reacted with the other antibody, indicating that individual molecules bearing both epitopes exist in brain. Peptide maps of the proteins immunoprecipitated with the two antibodies showed that they are closely similar. Despite these similarities, the two antibodies gave different staining patterns on sections of developing rat brain, anti-MAP5 staining both axons and dendrites whereas anti-MAP1x stained only axons. We conclude that the MAP5 and MAP1x molecules are very similar, and possibly identical. The difference in staining patterns with the two antibodies could be because there are two proteins present in brain, one in immature axons bearing both the MAP5 and MAP1x epitopes and another with a wider distribution bearing only the MAP5 epitope. Alternatively, there may be a single protein bearing both epitopes, with the MAP1x epitope being masked in neuronal dendrites and mature axons by covalent modification or inter-molecular binding.

Aging↗
PubMed1989

In situ localization of microtubule-associated protein mRNA in the developing and adult rat brain.

We have used cDNA probes specific for three of the major brain microtubule-associated proteins (MAPs), MAP1, MAP2, and MAP5, to study the timing of appearance, relative abundance, and intracellular compartmentalization of MAP gene transcripts in developing rat brain. The MAP1 probe hybridizes throughout the brain, in both grey and white matter. MAP2 mRNA is detected only in grey matter and appears in cerebral neurons only after they have ceased dividing and have migrated to the cortical plate. The MAP5 cDNA hybridizes throughout the embryonic brain, but by P12, MAP5 mRNA distribution is restricted to relatively immature areas. MAP2 mRNA, found in dendrites in the developing brain, persists in some adult dendrites. MAP5 mRNA, like beta-tubulin mRNA, is found only in the cell bodies of developing neurons, indicating that the protein must be transported from the soma into processes. MAP1 mRNA is found only in the proximal regions of cortical pyramidal cell dendrites in both developing and adult brain. The diverse distributions of MAP gene transcripts emphasize the importance of these proteins in generating heterogeneity of microtubule function and indicate that MAP compartmentalization within neurons is regulated in part by differential mRNA transport.

Aging↗
PubMed1988

Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites.

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.

Animals↗
PubMed1988

Single-shot cloning of multiple cDNAs coding for a set of related microtubule-associated proteins.

We describe a method for isolating multiple cDNA clones coding for a set of related proteins from bacteriophage lambda expression libraries in a single screening with polyclonal antiserum. The antiserum is raised against a tissue sub-fraction containing the proteins of interest; in the example presented this was brain microtubules. Each antibody-positive clone from the lambda expression library is plaque-purified and then grown in contact with nitrocellulose membrane that becomes coated with protein synthesized from the cloned cDNA. Each filter, containing the protein produced by a single lambda cDNA clone, is then used to affinity-select clone-specific antibodies from the original polyclonal antiserum. The monospecific antibody for each cDNA clone can be used on Western blots to identify the protein that the cDNA encodes and also to stain tissue sections. Using this method we have, in a single screening, obtained: (1) multiple cDNA clones representing different regions of a single large protein, (2) cDNA clones representing several functionally related proteins (microtubule-associated proteins), and (3) cDNA clones related to a novel protein species for which neither biochemical nor immunological data have previously been available.

Animals↗
PubMed1988

Different forms of microtubule-associated protein 2 are encoded by separate mRNA transcripts.

Brain microtubule-associated protein 2 (MAP2) consists of a pair of high molecular mass (280 kD) polypeptides, MAP2a and MAP2b, and a recently identified 70-kD protein, MAP2c, which is antigenically related to these high molecular mass MAP2's. Using cDNA clones we have analyzed the expression of these three proteins at the nucleic acid level. cDNA probes selective for the high molecular mass MAP2's a and b identified only a 9-kb mRNA, whereas a probe for sequence common to all three MAP2 isoforms, a, b, and c, recognized the 9-kb transcript and additionally a 6-kb mRNA. Southern blot analysis with cDNA probes indicated that there is only one MAP2 gene from which these two distinct mRNAs are derived. The 70-kD MAP2c protein is much more abundant in neurons of developing brain than those of adult tissues. Similarly the expression of the 6-kb MAP2c-related mRNA, is much greater in neonatal than adult rat brain, indicating that the developmental expression of MAP2 is determined by transcriptional regulation from a single MAP2 gene.

Animals↗
PubMed1988

A 70-kilodalton microtubule-associated protein (MAP2c), related to MAP2.

Microtubule-associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70-kilodalton protein that cross-reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70-kilodalton protein and MAP2 by peptide mapping. Our results show that the 70-kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the tau MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c-containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.

Adrenal Gland Neoplasms↗
PubMed1985

Operator mutations of the Escherichia coli aroF gene.

The Escherichia coli aroF and aroG genes encode the tyrosine-sensitive and the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, respectively, two of the three isoenzymes that control carbon flow through the shikimate pathway. Transcription of aroF and aroG is repressed by the tyrR gene product complexed to tyrosine or phenylalanine, respectively. Constitutive aroF mutants with lesions linked to aroF were isolated. The nucleotide sequences in the regulatory regions of aroF from six such mutants and from the parental wild type strain were determined. The mutations were found in two 18-base pair imperfect palindromes, called aroFo1 and aroFo2, which are located upstream of the aroF transcription start by 61 and 113 base pairs, respectively. Nuclease S1 mapping and analysis of in vitro run-off transcripts identified the 5'-end of the aroF transcript 51 base pairs upstream of the aroF translation start. The -35 region of the aroF promoter overlaps aroFo1. The aroFo1 and aroFo2 sequences are homologous to a single 18-base pair DNA segment preceding the coding sequence of aroG.

3-Deoxy-7-Phosphoheptulonate Synthase↗