David H. Gutmann

cDNA cloning of the type 1 neurofibromatosis gene : Complete sequence of the NF1 gene product

Von Recklinghausen neurofibromatosis, or type 1 neurofibromatosis (NF1), is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the embryonic neural crest. Portions of the gene have been recently identified by positional cloning, and sequence analysis has shown homology to the GTPase activating protein (GAP) family. In this report we present the results of an extensive cDNA walk resulting in the cloning of the complete coding region of the NF1 transcript. Analysis of the sequences reveals an open reading frame of 2818 amino acids, although alternatively spliced products may code for different protein isoforms. The gene extends for approximately 300 kb on chromosome 17, with its promoter in a CpG-rich island.

The neurofibromatosis type 1 gene and its protein product, neurofibromin

Von Recklinghausen neurofibromatosis, or neurofibromatosis type 1 (NFI), affects approximately 1 in 3500 individuals of all ethnic backgrounds. It is inherited as an autosomal dominant disease and is manifested clinically by abnormalities that predominantly affect tissues which derive from the neural crest (Riccardi, 1981,199l; Riccardi and Eichner, 1986). Affected individuals are often noted to have multiple cafeau-lait spots during the first year of life. These pigmented birthmarks contain melanocytes harboring macromelanosomes, but have noclinical significance other than as a diagnostic clue. Similarly, another important clinical feature, the presence of Lisch nodules of the iris, has no associated morbidity. However, these hamartomas appear during childhood and are eventually present in close to 100% of affected adults. Neurofibromas, which give the disease its name and usually make their appearance just before or during adolescence, are benign cutaneous tumors consisting of Schwann cells, fibroblasts, and other cellular elements. They increase in size and number with age, but at an unpredictable rate. More deeply placed neurofibromas, called plexiform lesions, usually appear in childhood and can lead to significant complications due to associated overgrowth of nearby tissues. Furthermore, such plexiform lesions have a modest but significant risk of degenerating into malignant neurofibrosarcoma, a highly invasive soft tissue tumor that is frequently fatal. This potential, plus the risk of optic glioma (which affects 2%-S% of all individuals with NFI) justifies placing this disease on the list of familial cancer syndromes (Bader, 1986). Other features of NFI arevariable but can be significant in a given patient. Approximately half of affected individuals have at least some degree of learning disability, and a small percentage have frank mental retardation (Riccardi, 1981). Seizures are present in about 5% of individuals, and megalencephaly is a typical finding. The diagnosis is usually not difficult to make in an adolescent or adult, but can occasionally present difficulties in a very young child. NFI is characterized by extreme variability, even among individuals within the same family who carry the same mutation. Approximately two-thirds of individuals with NFI lead relatively normal lives, with occasional interruptions for surgical management of their disease, often to remove neurofibromas that are causing cosmetic or physical distress. About one-third of individuals suffer a severe complication sometime during their lifetime. NFI has occasionally been erroneously referred to as “the elephant man disease”; while the gross facial distortion and other deformities present in Joseph Merrick, the Elephant Man, in some ways resemble the most severe end of the spectrum of NFI, subsequent evaluation of Merrick’s skeleton indicates that he probably had another disorder known as Proteus syndrome (Tibbles and Cohen, 1986).

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules

The neurofibromatosis type 1 (NF1) gene was recently identified by positional cloning and found to encode a protein with structural and functional homology to mammalian and yeast GTPase-activating proteins (GAPs). Using antibodies directed against the NF1 gene product, a protein of ∼250kDa was identified and termed neurofibromin. Double-indirect immunofluorescent labeling with anti-neurofibromin and anti-tubulin antibodies demonstrates that neurofibromin associates with cytoplasmic microtubules. Immunoblotting of microtubule-enriched cytoplasmic fractions, using antibodies generated against neurofibromin, shows that neurofibromin copurifies with microtubules. When portions of neurofibromin are expressed in Sf9 insect cells they associate with polymerized microtubules; furthermore, the critical residues for this interaction reside within the GAP-related domain of neurofibromin. The unexpected association of neurofibromin with microtubules suggests that neurofibromin is involved in microtubule-mediated intracellullar signal transduction pathways.

Parenthood: A Key to the Comparative Study of the Life Cycle

ABSTRACT Transcultural regularities in sex-role training, in the male and female response to the “chronic emergency” of parenthood, and in the phasing out of parental responsibilities all suggest that sex distinctions have an instrinsic basis, and that they are organized around the vital requirements of young children. It is further argued that parenthood constitutes the pivotal stage of the human life cycle, organizing the form and content of the stages which lead up to it, as well as those that succeed it. Accordingly, study of the common denominators and requirements of human parenthood could be an important first step in developing a comparative psychology of the human life cycle.

Personality changes associated with thalamic infiltration

SummaryMood and behavioral changes have been reported in patients with thalamic tumors. We report a case in which an infiltrating mass lesion of both thalami was manifested by an alteration in personality with relative motor and sensory sparing. These personality changes, unlike many such cases reported in the literature, were not related to increased intracranial pressure and hydrocephalus. Magnetic resonance imaging (MRI) demonstrated the lesion that was subtle and nearly inapparent on contrast-enhanced computerized tomography (CT).

In-vitro-derived bone marrow macrophages. Expression of Ia antigens during macrophage differentiation.

Cultures of bone marrow stem cells, grown in the presence of L-cell-conditioned medium, were harvested on successive days to determine the expression of Ia antigens and the acquisition of Ir4 gene-regulated antigen presentation. I-A subregion antigen expression was detected by an indirect radiobinding assay (RBA) as early as day 3 and reached maximal binding at day 7, before declining with additional time in culture. Indirect immunofluorescence (IIF) demonstrated 20% Ia+ cells on day 3 of culture and peaked at 60% on day 7 before declining with continued incubation. A mixed lymphocyte reaction (MLR) across an I region difference was used to assess the kinetics of bone-marrow-derived macrophage ( BMDM ) Ia expression. Maximal stimulation occurred with BMDM stimulator cells obtained from 5-9-day cultures. To investigate the acquisition of Ir gene function, BMDM harvested after various days in culture were used to reconstitute the T cell proliferative response to poly Glu60Ala30Tyr10 (GAT). The ability of BMDM to present GAT was detected after day 5, increased to maximal levels on day 7, and then declined with weak proliferative responses obtained using day 12 BMDM . The presentation of GAT by BMDM was inhibited by monoclonal anti-Ia. 17 antibody. Thus, the acquisition of Ir gene function by BMDM was found to parallel the expression of Ia molecules. Additional experiments were performed to determine whether treatment of BMDM at day 7 with lymphokines or beta-interferon could extend Ia antigen expression. Whereas treatment of day 7 BMDM cultures with Con-A-stimulated rat splenocyte supernatants extended and augmented Ia expression for an additional three days, beta-interferon treatment did not result in augmentation or extension of Ia antigen expression.

Lipid composition and in vitro biosynthetic rates of neutral lipids and phosphatidylcholine in anterior and posterior chambers of the goldfish swimbladder

Abstract 1. 1. Lipid composition was compared in anterior and posterior swimbladder and also in lavages of both chambers in the goldfish. 2. 2. Anterior bladder contained more phosphatidylethanolamine and sphingomyelin than posterior chamber, but phosphatidylcholine contents were similar. 3. 3. Cholesterol formed 17% of lipids in lavage-fluid in anterior chamber and 30% in posterior chamber. Lavage phospholipid contents in four pooled samples of each chamber were below detectable limits. Thus. surface-active phospholipids do not appear to be secreted by either chamber of goldfish swimbladder. 4. 4. Uptake and incorporation of [ 14 C]-acetate into total lipids, cholesterol and free fatty acids was greater in anterior than in posterior chamber. Rates of [ 3 H]methylcholine incorporation into phosphatidylcholine were similar in both chambers. 5. 5. Goldfish anterior and posterior swimbladder display differences in lipid composition and metabolism which may be related to functional specializations.

Major Histocompatibility Complex Regulation of the Immune Response

The ability of an organism to distinguish self from nonself is determined by a cluster of genes located in the major histocompatibility complex. Recent advances in molecular genetics and cellular immunology have begun to elucidate the mechanisms responsible for immune response regulation. In this review article, the genetic organization of the murine and human major histocompatibility complexes and the manner by which their gene products modulate immune responsiveness are discussed.

Characterization of three new intra-i region recombinant mouse strains, b10.asr7 (h-2as3), B10.bar4 (h-2h6), And b10.basr1 (h-2as4)

The ability to mount immune responses to T-dependent antigens is controlled by four genes that map to the I region of the murine major histocompatibility complex (McDevitt et al. 1981, Nagy et al. 1981). These Ir genes code for four distinct Ia polypeptide chains, As, Ap, E~, and E,, which associate to form the I-A (As; Ap) and I-E (E~;Ep) molecules (Uhr et al. 1979, Vitetta and Capra 1979). Restriction endonuclease analysis of the I region in H-2 recombinants with intra-I-region crossover events has demonstrated that tfiese recombinatorial events are concentrated in the area between the Ep and E= genes (Steinmetz et al. 1982, Hood et al. 1983). There are three previously described exceptions to this rule. D2.GD (H-2 °2) and A.TFR5 (H-2 apS) have intragenic recombinatorial events within the E~ gene (Jones 1981, Lafuse et al. 1980), while B10.STA62 has a recombinatorial event that separates the A~Ap duplex from the Ep gene (Singh et al. 1981). In this report, we describe two new H-2 haplotypes that very likely arose by recombination between the Ep locus and the A~A~ segment and one haplotype that may have arisen by recombination between the E~ and E~ loci. The current assignments of the I region alleles for B10.ASR7 (H-2as3), B10.BAR4 (H-2h6), and B10.BASR1 (//-2 a~4) were based on cytotoxicity data and indicated that in all three recombinant strains the crossover event occurred between the genes for H-2K and E~ (Klein et al. 1983). These three strains were then examined for expression of defined Ia specificities to determine whether the recombinant strain I-A molecule more closely resembled one parent or the other (Table 1). As determined by microcytotoxicity and confirmed by immunofluorescence (data not shown), the I-A molecules of all three strains exhibited monoclonal antibody binding patterns consistent with the k haplotype, in that 11.4.1 (H-2K k, m93), 11.5.2 (I-A k, m2), 10.2.16 (I-A k, I-A s, I-A f, m27), and 17/227 (I-A k, I-A b, I-A d, m15) bound, but MK-S4 (I-A s) and 28-16-8S (I-A b, I-A d, m8) failed to bind. Competitive inhibition radioimmunoassays performed to evaluate whether the