Cloning of a cDNA encoding the rat high molecular weight neurofilament peptide (NF-H): developmental and tissue expression in the rat, and mapping of its human homologue to chromosomes 1 and 22.
Neurofilaments (NFs) are the intermediate filaments specific to nervous tissue. They are probably essential to the tensile strength of the neuron, as well as to transport of molecules and organelles within the axon. Three peptides with apparent molecular masses of approximately 68 (NF-L), 145 (NF-M), and 200 (NF-H) kDa appear to be the major components of NF. The expression of these peptides is specific to nervous tissue and is developmentally regulated. Recently, complete cDNAs encoding NF-L and NF-M, and partial cDNAs encoding NF-H, have been described. To better understand the normal and pathophysiology of NFs we chose to clone the cDNA encoding the rat NF-H peptide. Using monoclonal antibodies that recognized NF-H, we screened a rat brain lambda gt11 library and identified a clone that contained a 2100-nucleotide cDNA insert representing the carboxyl-terminal portion of the NF-H protein. Anti-fusion protein antibodies recognized the NF-H peptide on immunoblots and stained fibrillar structures only in neurons. The cDNA recognized a 4500-nucleotide polyadenylated mRNA that was present only in nervous tissue and a 3500-nucleotide mRNA in adrenal. Brain NF-H mRNA levels were tightly developmentally regulated and paralleled the levels of NF-H peptide on immunoblots. Nuclear runoff studies showed that the 20-fold developmental increase in the NF-H message was due only in part to a 4-fold increase in its transcription rate. Levels of NF-H mRNA varied 20-fold among brain regions, with highest levels in pons/medulla, spinal cord, and cerebellum, and lowest levels in olfactory bulb and hypothalamus. Transcription studies revealed only a 2-fold difference in the transcription rates among these brain regions. Based on these results, we infer that half of the developmental increase and most of the interregional variation in the levels of the NF-H mRNA are mediated through message stabilization. Sequence information revealed that the carboxyl-terminal region of the NF-H peptide contained a unique serine-, proline-, alanine-, glutamic acid-, and lysine-rich repeat. The serine residues are likely sites of phosphorylation in the mature peptide. Genomic blots revealed a single copy of the gene in the rat genome and two copies in the human genome. In situ hybridizations performed on human chromosomes mapped the NF-H gene to chromosomes 1 and 22. Whether one copy is a pseudogene remains to be determined.
Lieberburg
I
I
Fishberg Center for Neurobiology, Mount Sinai School of Medicine, New York, NY 10029.
Spinner
N
N
Snyder
S
S
Anderson
J
J
Goldgaber
D
D
Smulowitz
M
M
Carroll
Z
Z
Emanuel
B
B
Breitner
J
J
Rubin
L
L
D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000196:embryology / Q000254:growth & development*; D002874:Chromosome Mapping; D002878:Chromosomes, Human, Pair 1; D002892:Chromosomes, Human, Pair 22; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005260:Female; D005796:Genes; D006801:Humans; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; C051774:neurofilament protein H; D004247:DNA