Thinning of large mammalian cells for cryo-TEM characterization by cryo-FIB milling.

Focused ion beam milling at cryogenic temperatures (cryo-FIB) is a valuable tool that can be used to thin vitreous biological specimens for subsequent imaging and analysis by cryo-transmission electron microscopy (cryo-TEM) in a frozen-hydrated state. This technique offers the potential benefit of eliminating the mechanical artefacts that are typically found with cryo-ultramicrotomy. However, due to the additional complexity in transferring samples in and out of the FIB, contamination and devitrification of the amorphous ice is commonly encountered. To address these problems, we have designed a sample cryo-shuttle that directly and specifically accepts Polara TEM cartridges to simplify the transfer process between FIB and TEM. We optimized several parameters in the cryo-FIB and cryo-TEM processes using the quality of the samples' ice as an indicator and demonstrated high-quality milling with large mammalian cells. By comparing the results from HeLa cells to those from Escherichia coli cells, we discuss some of the artefacts and challenges we have encountered using this technique.

Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos.

Homozygosity for the Egfr(tm1Mag) null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta were quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfr(tm1Mag) homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation.

Comparative genomic sequence analysis and isolation of human and mouse alternative EGFR transcripts encoding truncated receptor isoforms.

This study presents the annotated genomic sequence and exon-intron organization of the human and mouse epidermal growth factor receptor (EGFR) genes located on chromosomes 7p11.2 and 11, respectively. We report that the EGFR gene spans nearly 200 kb and that the full-length 170-kDa EGFR is encoded by 28 exons. In addition, we have identified two human and two mouse alternative EGFR transcripts of 2.4-3.0 kb using both computational and experimental methods. The human 3.0-kb and mouse 2.8-kb EGFR mRNAs are predominantly expressed in placenta and liver, respectively, and both transcripts encode 110-kDa truncated receptor isoforms containing only the extracellular ligand-binding domain. We also have demonstrated that the aberrant 2.8-kb EGFR transcript produced by the human A431 carcinoma cell line is generated by splicing to a recombinant 3'-terminal exon located in EGFR intron 16, which apparently was formed as a result of a chromosomal translocation. Finally, we have shown that the human, mouse, rat, and chicken 1.8- to 3.0-kb alternative EGFR transcripts are generated by distinct splicing mechanisms and that each of these mRNAs contains unique 3' sequences that are not evolutionarily conserved. The presence of truncated receptor isoforms in diverse species suggests that these proteins may have important functional roles in regulating EGFR activity.

Distinct signal/response mechanisms regulate pax1 and QmyoD activation in sclerotomal and myotomal lineages of quail somites.

Pax1 and QmyoD are early sclerotome and myotome-specific genes that are activated in epithelial somites of quail embryos in response to axial notochord/neural tube signals. In situ hybridization experiments reveal that the developmental kinetics of activation of pax1 and QmyoD differ greatly, suggesting that myotome and sclerotome specification are controlled by distinct developmental mechanisms. pax1 activation always occurs in somite IV throughout development, indicating that pax1 regulation is tightly coordinated with early steps in somite maturation. In contrast, QmyoD is delayed and does not occur until embryos have 12-14 somites. At this time, QmyoD is the first of the myogenic regulatory factor (MRF) genes to be activated in preexisting somites in a rapid, anterior to posterior progression until the 22 somite stage, after which time QmyoD is activated in somite I immediately following somite formation. Experiments involving transplantation of newly formed somites to ectopic sites along the anterior to posterior embryonic axis were performed to distinguish the contributions of axial signals and somite response pathways to the developmental regulation of pax1 and QmyoD. These studies show that pax1 activation is regulated by somite formation and maturation, not by the availability of axial signals, which are expressed prior to somite formation. In contrast, the delayed activation of QmyoD is controlled by developmental regulation of the production of axial signals as well as by the competence of somites to respond to these signals. These somite transplantation studies, therefore, provide a basis for understanding the different developmental kinetics of activation of pax1 and QmyoD during sclerotome and myotome specification, and suggest specific molecular models for the developmental regulation of myotome and sclerotome formation in somites through distinct signal/response pathways.

Notochord signals control the transcriptional cascade of myogenic bHLH genes in somites of quail embryos.

Microsurgical, tissue grafting and in situ hybridization techniques have been used to investigate the role of the neural tube and notochord in the control of the myogenic bHLH genes, QmyoD, Qmyf5, Qmyogenin and the cardiac alpha-actin gene, during somite formation in stage 12 quail embryos. Our results reveal that signals from the axial neural tube/notochord complex control both the activation and the maintenance of expression of QmyoD and Qmyf5 in myotomal progenitor cells during the period immediately following somite formation and prior to myotome differentiation. QmyoD and Qmyf5 expression becomes independent of axial signals during myotome differentiation when somites activate expression of Qmyogenin and alpha-actin. Ablation studies reveal that the notochord controls QmyoD activation and the initiation of the transcriptional cascade of myogenic bHLH genes as epithelial somites condense from segmental plate mesoderm. The dorsal medial neural tube then contributes to the maintenance of myogenic bHLH gene expression in newly formed somites. Notochord grafts can activate ectopic QmyoD expression during somite formation, establishing that the notochord is a necessary and sufficient source of diffusible signals to initiate QmyoD expression. Myogenic bHLH gene expression is localized to dorsal medial cells of the somite by inhibitory signals produced by the lateral plate and ventral neural tube. Signaling models for the activation and maintenance of myogenic gene expression and the determination of myotomal muscle in somites are discussed.

Inhibition of proliferation of human melanocytes by a KIT antisense oligodeoxynucleotide: implications for human piebaldism and mouse dominant white spotting (W).

KIT constitutes the cell surface transmembrane receptor protein tyrosine kinase for a growth factor variously termed steel factor (SLF), stem cell factor, mast cell growth factor, or Kit ligand. Inherited mutations of the KIT gene result in piebaldism in humans and dominant white spotting (W) in mice. Patches of hypopigmented skin and hair in these disorders represent regions lacking in melanocytes, the result of defective melanoblast differentiation, migration, proliferation, or survival during embryonic development. Here we show that incubation of normal human melanocytes with a KIT antisense oligodeoxynucleotide greatly inhibits cell proliferation in culture, whereas incubation with a KIT sense oligodeoxynucleotide has no effect. The KIT oligodeoxynucleotides also had little or no effect on cell survival.

A YAC contig spanning a cluster of human type III receptor protein tyrosine kinase genes (PDGFRA-KIT-KDR) in chromosome segment 4q12.

We have mapped five genes encoding protein tyrosine kinases (PTKs) to the pericentromeric region of human chromosome 4. PTK4 and TYRO4, which encode nonreceptor intracellular PTKs, are located at 4p12 and 4q13, respectively. The other three genes, PDGFRA, KIT, and KDR, encode type III transmembrane receptor PTKs for known ligands. We have developed a contig of 29 yeast artificial chromosomes (YACs) spanning approximately 2 Mb of DNA at 4q12 that includes PDGFRA, KIT, and KDR, and we have used this YAC contig to map 12 different sequence-tagged sites in this region. PDGFRA, KIT, and KDR thus constitute a cluster of genes at 4q12 encoding closely related type III receptor PTKs. Mutations of the human KIT gene result in piebaldism, an autosomal dominant disorder of melanocyte development.

PTK1, a novel protein kinase required for proliferation of human melanocytes.

We have identified and characterized PTK1, a novel human non-receptor protein kinase. Partial PTK1 cDNA was initially isolated by RT-PCR from mRNA of normal human melanocytes. Northern blot hybridization detected a 3.8-kb PTK1 mRNA in all tissues and cell types studied. Nucleotide sequence analysis of a full-length PTK1 cDNA showed it to encode an 847-amino acid polypeptide with an amino-terminal SH3 domain, a kinase catalytic domain, a leucine zipper-like domain, and a carboxyl proline-rich domain. The PTK1 kinase domain contains motifs generally considered diagnostic of serine/threonine kinases but also contains amino acids highly conserved among the tyrosine kinases. This suggests that PTK1 is a serine/threonine kinase, but one very closely related to the tyrosine kinase superfamily. When normal human melanocytes were exposed to antisense PTK1 oligonucleotide, cell growth was inhibited, whereas a corresponding sense oligonucleotide had no inhibitory effect. These data indicate that PTK1 plays an important role in the proliferation of normal human melanocytes.

A survey of protein tyrosine kinase mRNAs expressed in normal human melanocytes.

We have used the reverse transcription-polymerase chain reaction to survey the repertoire of protein tyrosine kinases expressed in cultured normal human melanocytes, a differentiated cell type derived from the neural crest. We identified 25 different tyrosine kinase cDNAs among a total of 608 protein tyrosinase kinase-related cDNAs analyzed. Six encode receptor tyrosine kinases for known ligands, several of which have been implicated in controlling melanocyte proliferation in vitro. Two others encode apparent receptor tyrosine kinases for unknown ligands. Four encode known non-receptor tyrosine kinases and five encode previously identified anonymous protein tyrosine kinases. Of the eight other melanocyte-associated protein tyrosine kinases, most or all appear to be novel. These 25 protein tyrosine kinase genes exhibit distinct patterns of expression in cultured human melanocytes, human erythroleukemia cells, and a variety of normal human tissues. We mapped 16 of the corresponding protein tyrosine kinase genes to specific human chromosomes, identifying a total of 19 human genetic loci, some of which may constitute candidate genes for genetic disorders of mammalian development.

Mutations of the tyrosinase gene in Indo-Pakistani patients with type I (tyrosinase-deficient) oculocutaneous albinism (OCA).

Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by deficient synthesis of melanin pigment. Type I (tyrosinase-deficient) OCA results from mutations of the tyrosinase gene (TYR gene) encoding tyrosinase, the enzyme that catalyzes the first two steps of melanin biosynthesis. Mutations of the TYR gene have been identified in a large number of patients, most of Caucasian ethnic origin, with various forms of type I OCA. Here, we present an analysis of the TYR gene in eight Indo-Pakistani patients with type I OCA. We describe four novel TYR gene mutations and a fifth mutation previously observed in a Caucasian patient.

A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism.

The mouse pink-eyed dilution (p) locus on chromosome 7 is associated with defects of skin, eye and coat pigmentation. Mutations at p cause a reduction of eumelanin (black-brown) pigment and altered morphology of black pigment granules (eumelanosomes), but have little effect on pheomelanin (yellow-red) pigment. We show here that the human complementary DNA DN10, linked to the p locus in mice, identifies the human homologue (P) of the mouse p gene, and appears to encode an integral membrane transporter protein. The expression pattern of this gene in various p mutant mice correlates with the pigmentation phenotype; moreover, an abnormally sized messenger RNA is detected in one mutant, p(un), which reverts to the normal size in p(un) revertants. The human P gene corresponds to the D15S12 locus within the chromosome segment 15q11-q13, which is typically deleted in patients with Prader-Willi and Angelman syndrome (see ref. 5 for review). These disorders are phenotypically distinct, depending on the parent of origin of the deleted chromosome, but both syndromes are often associated with hypopigmentation of the skin, hair and eyes (see ref. 8 for review), and deletion of the P gene may be responsible for this hypopigmentation. In addition, we report a mutation in both copies of the human P gene in one case of tyrosinase-positive (type II) oculocutaneous albinism, recently linked to 15q11-q13 (ref. 9).

Organization and nucleotide sequence of the human KIT (mast/stem cell growth factor receptor) proto-oncogene.

We have cloned and sequenced the human KIT proto-oncogene, which contains 21 exons and spans more than 34 kb of DNA on chromosome segment 4q12. We also establish physical linkage between the KIT gene and the related PDGFRA gene. The organization of the KIT gene is virtually identical to that of the homologous FMS gene, located on chromosome 5. Together, these data suggest that the KIT and PDGFRA genes on chromosome 4 and the FMS and PDGFRB genes on chromosome 5 arose by duplication of a common ancestral gene, followed by duplication of a chromosome.

Tyrosinase gene mutations in type I (tyrosinase-deficient) oculocutaneous albinism define two clusters of missense substitutions.

Type I (tyrosinase-deficient) oculocutaneous albinism (OCA) results from mutations of the gene encoding tyrosinase, the enzyme that catalyzes the first 2 steps of melanin pigment biosynthesis. In type IA (tyrosinase-negative) OCA tyrosinase enzymatic activity is completely absent, and in type IB ("yellow") OCA tyrosinase activity is greatly reduced. Here, we describe 11 novel mutations of the tyrosinase gene in Caucasian patients with these 2 forms of type I OCA. Type I OCA in Caucasians appears to result from a great variety of different uncommon alleles. More than 80% of the known missense substitutions associated with type I OCA cluster within 2 relatively small regions of the tyrosinase polypeptide, suggesting that these may correspond to functionally important sites within the enzyme.

Tyrosinase gene mutations associated with type IB ("yellow") oculocutaneous albinism.

We have identified three different tyrosinase gene mutant alleles in four unrelated patients with type IB ("yellow") oculocutaneous albinism (OCA) and thus have demonstrated that type IB OCA is allelic to type IA (tyrosinase negative) OCA. In an inbred Amish kindred, type IB OCA results from homozygosity for a Pro----Leu substitution at codon 406. In the second family, type IB OCA results from compound heterozygosity for a type IA OCA allele (codon 81 Pro----Leu) and a novel type IB allele (codon 275 Val----Phe). In the third patient, type IB OCA results from compound heterozygosity for the same type IB allele (codon 275 Val----Phe) and a novel type IB OCA allele. In a fourth patient, type IB OCA results from compound heterozygosity for the codon 81 type IA OCA allele and a type IB allele that contains no identifiable abnormalities; dysfunction of this type IB allele apparently results from a mutation either well within one of the large introns or at some distance from the tyrosinase gene. In vitro expression of the Amish type IB allele in nonpigmented HeLa cells demonstrates that the Pro----Leu substitution at codon 406 greatly reduces but does not abolish tyrosinase enzymatic activity, a finding consistent with the clinical phenotype.

A polymorphism of the human tyrosinase gene is associated with temperature-sensitive enzymatic activity.

We have identified a common nonpathological polymorphism of the human tyrosinase gene. In Caucasians codon 402 can be either CGA (arginine) [p = .85] or CAA (glutamine) [p = .15]. This polymorphism also occurs in American Blacks, but the codon 402CAA (Gln) allele was not detected in Oriental populations. The substitution of glutamine for arginine at codon 402 results in moderate thermoinstability of the corresponding tyrosinase polypeptide. Tyrosinase enzymatic activity expressed in HeLa cells transfected with a codon 402Gln tyrosinase cDNA is reduced by approximately 75 percent when cells are cultured at 37 degrees C as compared to 31 degrees C, whereas enzymatic activity of codon 402Arg tyrosinase is not temperature-sensitive. However, the genotype at codon 402 of tryosinase is not correlated with the apparent pigmentation phenotype in normal Caucasians.

Organization and nucleotide sequences of the human tyrosinase gene and a truncated tyrosinase-related segment.

We have isolated and sequenced the gene encoding human tyrosinase, the key enzyme in pigment biosynthesis. The human tyrosinase gene contains five exons and spans more than 50 kb of DNA on chromosome segment 11q14----q21. We have also isolated a second segment in the human genome that is closely related to tyrosinase. The tyrosinase-related segment, located on 11p11.2----cen, contains only exons 4 and 5 plus adjacent noncoding regions. This segment is present in all human ethnic groups analyzed, and the noncoding nucleotide sequences shared by the 11q tyrosinase gene and the 11p tyrosinase-related segment differ by only 2.6%. This suggests that this segment of the tyrosinase gene was duplicated approximately 24 million years ago.

Homozygous tyrosinase gene mutation in an American black with tyrosinase-negative (type IA) oculocutaneous albinism.

We have identified a tyrosinase gene mutation in an American black with classic, tyrosinase-negative oculocutaneous albinism. This mutation results in an amino acid substitution (Cys----Arg) at codon 89 of the tyrosinase polypeptide. The proband is homozygous for the substitution, suggesting that this mutation may be frequently associated with tyrosinase-negative oculocutaneous albinism in blacks.

Human U1-70K ribonucleoprotein antigen gene: organization, nucleotide sequence, and mapping to locus 19q13.3.

We have isolated and sequenced the gene encoding the human U1-70K snRNP protein. U1-70K is an RNA-binding protein that is a specific component of the U1 small nuclear ribonucleoprotein complex (snRNP) and constitutes the major anti-(U1) RNP autoimmune antigen. We have mapped the U1-70K gene to the distal portion of chromosome 19, at band q13.3. The gene is greater than 44 kb in size and consists of 11 exons. The general structure of the gene has been completely conserved during vertebrate evolution and accounts for the production of several different U1-70K mRNA species by alternative pre-mRNA splicing. Comparison of the predicted amino acid sequences of animal U1-70K proteins reveals a high degree of conservation, particularly in the region of the RNP consensus domain. Even more striking is the complete conservation of the nucleotide sequence of an alternative included/excluded exon containing an in-frame translational termination codon. This conservation also includes significant portions of the downstream intervening sequence. This extraordinary conservation at the nucleotide sequence level suggests that alternative splicing of this exon serves an important function, perhaps in regulating the production of functional U1-70K protein.