Sequence and structure of the mouse gene coding for the largest neurofilament subunit

We have determined the complete nucleotide sequence of the mouse gene encoding the neurofilament NF-H protein. The C-terminal domain of NF-H is very rich in charged amino acids (aa) and contains a 3-aa sequence, Lys-Ser-Pro, that is repeated 51 times within a stretch of 368 aa. The location of this serine-rich repeat in the phosphorylated domain of NF-H indicates that it represents the major protein kinase recognition site. The nfh gene shares two common intron positions with the nfl and nfm genes, but has an additional intron that occurs at a location equivalent to one of the introns in non-neuronal intermediate filament-coding genes. This additional nfh intron may have been acquired via duplication of a primordial intermediate filament gene.     

Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M

We have generated a set of amino- and carboxy-terminal deletions of the neurofilament NF-M gene and determined the molecular consequences of forced expression of these mutant constructs in mouse fibroblasts. To follow the expression of mutant NF-M subunits in transfected cells, a 12 amino acid epitope (from the human c-myc protein) was expressed at the carboxy terminus of each mutant. We show that NF-M molecules missing up to 90 or 70% of the nonhelical carboxy-terminal tail or amino-terminal head domains, respectively, incorporate readily into an intermediate filament network comprised either of vimentin or NF-L, whereas deletions into either the amino- or carboxy-terminal alpha- helical rod region generate assembly-incompetent polypeptides. Carboxy- terminal deletions into the rod domain invariably yield dominant mutants which rapidly disrupt the array of filaments comprised of NF-L or vimentin. Accumulation of these mutant NF-M subunits disrupts vimentin filament arrays even when present at approximately 1% the level of the wild-type subunits. In contrast, the amino-terminal deletions into the rod produce pseudo-recessive mutants that perturb the wild-type NF-L or vimentin arrays only modestly. The inability of such amino-terminal mutants to disrupt wild-type subunits defines a region near the amino-terminal alpha-helical rod domain (residues 75- 126) that is required for the earliest steps in filament assembly.     

Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein

We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked.     

The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it.

Genomic clones for the largest human neurofilament protein (NF-H) were isolated, the intron/exon boundaries mapped and the entire protein-coding regions (exons) sequenced. The predicted protein contains a central region that obeys the structural criteria identified for alpha-helical 'rod' domains typically present in all IF protein components: it is approximately 310 amino acids long, shares amino acid sequence homology with other IF protein rod domains and displays the characteristic heptad repeats of apolar amino acids which facilitate coiled-coil interaction. Nevertheless, anomalies are noted in the structure of the NF-H rod which could explain observations of its poor homopolymeric assembly in vitro. The protein segment on the carboxy-terminal side of the human NF-H rod is uniquely long (greater than 600 amino acids) compared to other IF proteins and is highly charged (greater than 24% Glu, greater than 25% Lys), rich in proline (greater than 12%) and impoverished in cysteine, methionine and aromatic amino acids. Its most remarkable feature is a repetitive sequence that covers more than half its length and includes the sequence motif, Lys-Ser-Pro (KSP) greater than 40 times. Together with the recent identification of the serine in KSP as the main target for NF-directed protein kinases in vivo, this repetitive character explains the massive phosphorylation of the NF-H subunit that can occur in axons. The human NF-H gene has three introns, two of which interrupt the protein-coding sequence at identical points to introns in the genes for the two smaller NF proteins, NF-M and NF-L.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-chemical characterization of NF-H, the largest mammalian neurofilament component; intermediate filament-type sequences followed by a unique carboxy-terminal extension

NF-H has the highest mol. wt. of the three mammalian neurofilament components (NF-L, NF-M, NF-H). In spite of its unusually large mol. wt., estimated to be 200 K by gel electrophoresis, NF-H contains sequences which identify it as an integral intermediate filament (IF) protein in its amino-terminal region. We have isolated and partially characterized a basic, non-α-helical segment located at the amino-terminal end with properties similar to headpieces of other non-epithelial IF proteins. The highly α-helical 40-K fragment excised by chymotrypsin is now identified by the amino acid sequence of a 17-K fragment. This sequence can be unambiguously aligned with the rod region of other IF proteins and covers about half of the presumptive coiled-coil arrays. NF-H and NF-M show 45% sequence identity in this region. The extra mass of NF-H in comparison with most other IF proteins arises from a carboxy-terminal extension thought to be responsible for inter-neurofilament cross-bridges in axons. This autonomous domain has a unique amino acid composition characterized by a high content of proline, alanine and particularly of lysine and glutamic acid. The NF-H tailpiece extension also carries a large number of serine phosphates, which are not evenly distributed, but are restricted to the amino-terminal part. Having now delineated the intermediate filament-type sequences for all three neurofilament proteins it seems very likely that the three components interact via coiled-coil interactions. They all carry unique carboxy-terminal extensions which increase in length from NF-L to NF-H and seem to extend from the filament wall.     

Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum.

The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence.     

Complete sequence of a bovine type I cytokeratin gene: conserved and variable intron positions in genes of polypeptides of the same cytokeratin subfamily

The complete sequence of a bovine gene encoding an epidermal cytokeratin of mol. wt. 54 500 (No VIb) of the acidic (type I) subfamily is presented, including an extended 5' upstream region. The gene (4377 bp, seven introns) which codes for a representative of the glycine-rich subtype of cytokeratins of this subfamily, is compared with genes coding for: another subtype of type I cytokeratin; a basic (type II) cytokeratin gene; and vimentin, a representative of another intermediate filament (IF) protein class. The positions of the five introns located within the highly homologous alpha-helix-rich rod domain are identical or equivalent, i.e., within the same triplet, in the two cytokeratin I genes. Four of these intron positions are also identical with intron sites in the vimentin gene, and three of these intron positions are identical or similar in the type I and type II cytokeratin subfamilies. On the other hand, the gene organization of both type I cytokeratins differs from that of the type II cytokeratin in the rod region in five intron positions and in the introns located in the carboxy-terminal tail region, with the exception of one position at the rod-tail junction. Remarkably, the two type I cytokeratins also differ from each other in the positions of two introns located at and in the region coding for the hypervariable, carboxy-terminal portion. The introns and the 5' upstream regions of the cytokeratin VIb gene do not display notable sequence homologies with the other IF protein genes, but sequences identical with--or very similar to--certain viral and immunoglobulin enhancers have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the mouse glial fibrillary acidic protein gene: implications for the evolution of the intermediate filament multigene family.

We report the complete sequence of the gene encoding mouse glial fibrillary acidic protein (GFAP), the intermediate filament (IF) protein specific to astrocytes. The 9.8 kb gene includes nine exons separated by introns ranging in size from 0.2 to 2.5 kb. A comparison of the organization of the GFAP gene with that of genes encoding other IF proteins reveals that the structure of IF genes is highly conserved in spite of considerable divergence at the amino acid level. Thus, most of the evolutionary events leading to the placement of introns in IF genes must have occurred prior to the duplication and subsequent divergence of IF genes from a presumptive common ancestral sequence. The conserved gene organization is unrelated to structural features of IF proteins. A curious feature of the GFAP gene is the large number of repeated sequences found in the introns. Six tracts of reiterated di- or trinucleotides are present, plus tandem repeats of two different novel sequences. One repeat is unique to the GFAP gene; the other occurs elsewhere in the mouse genome, although at relatively low frequency.     

Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha- internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.     

Assembly of carboxy-terminally deleted desmin in vimentin-free cells.

Using a vimentin-free expression system we were able to demonstrate that the carboxy terminus of desmin is necessary for filament assembly in the living cell. Desmin subunits missing only 4 carboxy-terminal residues of their rod domain are incapable of homopolymeric filament assembly. Moreover, even single amino acid substitutions in the conserved carboxy-terminal part of the rod domain prevent desmin subunits from homopolymeric filament assembly. Desmin subunits missing 18 or more carboxy-terminal residues of their rod domain (including the complete conserved carboxy-terminal region) are unstable in cells devoid of intact type III intermediate filaments (IFs). Interaction with an intact type III IF, however, stabilizes these mutated desmin subunits. Expression of a desmin subunit missing both its non-helical end domains in vimentin-containing cells disrupts the endogenous vimentin network completely.     

Developmentally regulated cytokeratin gene in Xenopus laevis.

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.     

Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension

The sequence of the amino-terminal 436 residues of porcine neurofilament component NF-M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non-epithelial type. A non-alpha-helical arginine-rich headpiece with multiple beta-turns (residues 1-98) precedes a highly alpha-helical rod domain able to form double-stranded coiled-coils (residues 99-412) and a non-alpha-helical tailpiece array starting at residue 413. All extra mass of NF-M forms, as a carboxy-terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino-terminal region of this domain document a lysine- and particularly glutamic acid-rich array somewhat reminiscent of the much shorter tailpiece extension of NF-L (apparent mol. wt. 68 kd), the major neurofilament protein. NF-M is therefore a true intermediate filament protein co-polymerized with NF-L via presumptive coiled-coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF-L. Along the structurally conserved coiled-coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF-L and NF-M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra-filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.     

The chicken vimentin gene. Nucleotide sequence, regulatory elements, and comparison to the hamster gene

Here we report the nucleotide sequence of the chicken vimentin gene and its deduced primary amino acid sequence. A comparison of this gene to other intermediate filament protein genes demonstrates that both exon size and position are strongly conserved features of this multigene family. In addition, the hamster and chicken vimentin genes exhibit strong identity at the level of nucleotide (74%) and amino acid (80%) sequence. Interestingly, 40% of total sequence diversity is localized to the N terminus or "head" region of these genes whereas other protein domains (rod and C terminus) are remarkably identical in both nucleotide (81%) and amino acid (89%) sequence. Even stronger amino acid identity (100%) is exhibited in certain subdomains which may define regions crucial for filament formation and function. Not surprisingly, vimentin is more homologous across animal species than it is to other intermediate filament protein members (e.g. desmin) within the same species. A comparison of 5'-flanking sequences of the hamster and chicken genes as well as other characterized promoter elements (SV40, HSV-TK) reveals homologous sequence elements which may define common and/or unique sites involved in the modulation of gene expression. The implications of these sequence elements for both tissue-specific and developmental expression of the vimentin gene are discussed.     

Nucleotide sequence of the gene for the b subunit of human factor XIII

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.