Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: Termopsidae)

Although “polymorphic castes” in social insects are well known as one of the most important phenomena of polyphenism, few studies of caste-specific gene expressions have been performed in social insects. To identify genes specifically expressed in the soldier caste of the Japanese damp-wood termite Hodotermopsis japonica, we employed the differential-display method using oligo(dT) and arbitrary primers, compared mRNA from the heads of mature soldiers and pseudergates (worker caste), and identified a clone (PCR product) 329 bp in length termed SOL1. Northern blot analysis showed that the SOL1 mRNA is about 1.0 kb in length and is expressed specifically in mature soldiers, but not in pseudergates, even in the presoldier induction by juvenile hormone analogue, suggesting that the product is specific for terminally differentiated soldiers. By using the method of 5′- and 3′-rapid amplification of cDNA ends, we isolated the full length of SOL1 cDNA, which contained an ORF with a putative signal peptide at the N terminus. The sequence showed no significant homology with any other known protein sequences. In situ hybridization analysis showed that SOL1 is expressed specifically in the mandibular glands. These results strongly suggest that the SOL1 gene encodes a secretory protein specifically synthesized in the mandibular glands of the soldiers. Histological observations revealed that the gland actually develops during the differentiation into the soldier caste.

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2b mice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2b mouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.

Quantitative insight into proliferation and differentiation of oligodendrocyte type 2 astrocyte progenitor cells in vitro

As part of our attempts at understanding fundamental principles that underlie the generation of nondividing terminally differentiated progeny from dividing precursor cells, we have developed approaches to a quantitative analysis of proliferation and differentiation of oligodendrocyte type 2 astrocyte (O-2A) progenitor cells at the clonal level. Owing to extensive previous studies of clonal differentiation in this lineage, O-2A progenitor cells represent an excellent system for such an analysis. Previous studies have resulted in two competing hypotheses; one of them suggests that progenitor cell differentiation is symmetric, the other hypothesis introduces an asymmetric process of differentiation. We propose a general model that incorporates both such extreme hypotheses as special cases. Our analysis of experimental data has shown, however, that neither of these extreme cases completely explains the observed kinetics of O-2A progenitor cell proliferation and oligodendrocyte generation in vitro. Instead, our results indicate that O-2A progenitor cells become competent for differentiation after they complete a certain number of critical mitotic cycles that represent a period of symmetric development. This number varies from clone to clone and may be thought of as a random variable; its probability distribution was estimated from experimental data. Those O-2A cells that have undergone the critical divisions then may differentiate into an oligodendrocyte in each of the subsequent mitotic cycles with a certain probability, thereby exhibiting the asymmetric type of differentiation.

Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.

The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (mda-7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda-7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda-7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda-7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda-7, Ad.mda-7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.mda-7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda-7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda-7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda-7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.

Bacterial diversity within the human subgingival crevice

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.

Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.

Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells

Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using blast searches against dbest for FAD-, NAD(P)H-binding sequences followed by reverse transcription–PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome c reduction in vitro. The properties of this protein make it a plausible candidate oxygen sensor.

Mice cloned from embryonic stem cells

Cloning allows the asexual reproduction of selected individuals such that the offspring have an essentially identical nuclear genome. Cloning by nuclear transfer thus far has been reported only with freshly isolated cells and cells from primary cultures. We previously reported a method of cloning mice from adult somatic cells after nuclear transfer by microinjection. Here, we apply this method to clone mice from widely available, established embryonic stem (ES) cell lines at late passage. With the ES cell line R1, 29% of reconstructed oocytes developed in vitro to the morula/blastocyst stage, and 8% of these embryos developed to live-born pups when transferred to surrogate mothers. We thus cloned 26 mice from R1 cells. Nuclei from the ES cell line E14 also were shown to direct development to term. We present evidence that the nuclei of ES cells at G1- or G2/M-phases are efficiently able to support full development. Our findings demonstrate that late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period.

Identification of transforming growth factor-β- regulated genes in Caenorhabditis elegans by differential hybridization of arrayed cDNAs

Members of the transforming growth factor-β family play critical roles in body patterning, in both vertebrates and invertebrates. One transforming growth factor-β-related gene, dbl-1, has been shown to regulate body length and male ray patterning in Caenorhabditis elegans. We screened arrayed cDNAs to identify downstream target genes for the DBL-1 signaling by using differential hybridization. C. elegans cDNAs representing 7,584 independent genes were arrayed on a nylon membrane at high density and hybridized with 33P-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2, and lon-2 worms. Signals for all the spots representing hybridized DNA were quantified and compared among strains. The screening identified 22 and 2 clones, which were positively and negatively regulated, respectively, by the DBL-1 signal. Northern hybridization confirmed the expression profiles of most of the clones, indicating good reliability of the differential hybridization using arrayed cDNAs. In situ hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed that at least four genes, including the gene for the type I receptor for DBL-1, sma-6, were transcriptionally regulated by the DBL-1 signal.

Identification of a structural determinant for resistance to β-lactam antibiotics in Gram-positive bacteria

Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment. Clinical resistance of S. pneumoniae to β-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for β-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple β-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the “sensitive” form of PBP2x produces mutants similar, in terms of β-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for β-lactams and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H-PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivity to therapeutic concentrations of β-lactam antibiotics is lost.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Mosquito transferrin, an acute-phase protein that is up-regulated upon infection

When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both Aedes aegypti and Aedes albopictus. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an A. aegypti transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3′ end exactly matched a cDNA encoding a polypeptide that is up-regulated when A. aegypti encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994) Exp. Parasitol. 79, 312–321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron.