Medium weight neurofilament mRNA in goldfish Mauthner axoplasm

Abstract

Although axons are generally considered to lack the ability to synthesize proteins, the Mauthner axon (M-axon) of the goldfish has been reported to contain some of the basic components of the translational machinery, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and ribosomes. To determine if the M-axon also contains mRNA, we isolated samples of M-axoplasm free of glial contamination as demonstrated by the absence of glial-specific mRNA and protein. Reverse transcription-polymerase chain reaction (RT-PCR) of M-axoplasmic cDNA in the presence of primers for the goldfish medium-weight neurofilament (NF-M) gene produced a single product of the expected length for RT-PCR amplification of goldfish NF-M mRNA. This mRNA might direct protein synthesis of NF-M within the M-axoplasm.

Fig. 1

Location and sequence of primers used in PCR amplifications. (A) Goldfish NF-M gene. The horizontal lines indicate location of introns whereas the boxes indicate the location of exons for the goldfish NF-M gene. The location of the sense and antisense primers used in PCR amplifications are denoted by forward and reverse half-arrows. Sense and antisense primers correspond to nucleotides 1716–1740 and 3119–3243, respectively, of the goldfish NF-M gene [8]. (B) GFAP gene. Intron/exon structure and primer designations are as described in (A). The location and size of introns for the goldfish GFAP gene were inferred by comparing the sequence of goldfish GFAP eDNA (Glasgow and Schechter, unpublished; Genbank Accession Number L23876) with the published sequence for the mouse GFAP gene [1]. The application of this method to compare the goldfish NF-M gene [8] with the mouse NF-M gene [17] showed that intron location, but not intron size, was conserved between goldfish and mouse. Sense and antisense primers correspond to nucleotides 648–672 and 975–999, respectively, of goldfish GFAP eDNA (Glasgow and Schechter, unpublished; Genbank Accession Number L23876).

Fig. 2

(A) Products of PCR amplification of NF-M mRNA in goldfish brain and M-axoplasm. Total eDNA prepared from adult brain (lane 1) and M-axoplasm (lane 2) was subjected to 35 cycles of PCR amplification using forward and reverse NF-M primers. Lane 3 contaitts the amplification product of the reagents for NF-M PCR amplification without the inclusion of eDNA derived from goldfish tissue. Each lane contains 20% of a total PCR amplification. The arrow labeled NF-M indicates the theoretical location of the PCR product for NF-M mRNA at 387 nucleotides. The numbers to the left of (A,B) indicate the nucleotide length of Hpall cut pUCI9 marker DNA. (B) Products of PCR amplification of GFAP mRNA in goldfish brain and M-axoplasm. The same brain and M-axoplasmic eDNA samples shown in (A) were subjected to 35 cycles of PCR amplification using forward and reverse GFAP primers. Lane 3 contains the amplification product of the reagents for GFAP PCR amplification without the inclusion of eDNA derived from goldfish tissue. PCR products were analyzed as in (A). The arrow labeled GFAP indicates the theoretical location of the PCR product for GFAP mRNA at 352 nucleotides.

Fig. 3

Silver-stained gel and anti-GFAP immunoblot of M-axoplasm and M-sheath. Goldfish M-axoplasm and M-sheath were electrophoretically analyzed on 10 to 15% gradient denaturing polyacrylamide gels. The gels were either silver-stained or the proteins were transferred to a nitrocellulose filter and probed with monoclonal antibodies directed against GFAP. Lanes 1 and 4 contain silver-stained M-axoplasmic and M-sheath proteins, respectively. Lanes 2 and 3 contain anti-GFAP immunoblots of M-axoplasm and M-sheath, respectively. The lines to the left of the figure indicate the location of neurofilament proteins in M-axoplasm at 235, 145, 123, 105, 80, and 60 kDa. The arrow to the right of the figure indicates the location of GFAP in M-sheath at 50 kDa.