Gene Families

The following sections are included:

• PREFACE

• OBITUARY

• TABLE OF CONTENTS

Thank you Dr. VandeBerg, members and guests of the Chinese Academy of Sciences and participants in the Congress.

In April 1997, Clem Markert was diagnosed with carcinoma of the colon and underwent right hemi-colectomy. The pathology report showed 19 positive lymph nodes in a tumor that had invaded through the bowel serosa. He underwent three months of chemotherapy and was then discovered to have widespread pleural pulmonary metastases. For the next two years, he led a very active life with trips to Alaska and Africa, and boating on the Columbia and Snake Rivers. His course for the past three months, however, was one of steady deterioration - to the point that he knew he would be unable to attend the 10th International Congress. He died on the evening of October 1^st, 1999 in Colorado Springs…

A combination of EST analysis, application of bioinformatics, primer walking, reverse-transcription PCR and RACE has been widely used in obtaining novel full-length cDNAs. By applying this method we have cloned 600 novel full-length cDNAs sequences mainly from endocrine and hematopoietic systems. Some of these genes can be categorized into several gene families, which included some transcription factors and those involved in vesicle trafficking and signal transduction. There are also many novel genes showing homology to genes discovered in relatively lower creatures. The bioinformatic analysis combined with experimental methods were used for identifying new members of known gene superfamilies.

The mammalian testis is a unique organ programmed for both endocrine and germ cell production, functions that are clearly interdependent. The germ cell component displays distinctive developmental properties illustrated by programmed molecular events that occur with the onset and during spermatogenesis and include activation and inactivation of numerous genes yielding protein products with distinct or modified properties. Genes expressed during spermatogenesis can be classified as “housekeeping” or structural. Both categories include testis specific isozymes and isoforms. One such example is LDH-C₄, a member of the lactate dehydrogenase gene family that is transcribed only during prophase of the first meiotic division. We have cloned and sequenced the promoter of this gene and demonstrated functionality. Even though this gene and protein are well-studied, there remains the question of why LDH-C₄ supplants the other lactate dehydrogenases in testis and sperm metabolism.

A second example of an unique protein is provided by calpastatin. This protein is the endogenous inhibitor of calpain, a cytoplasmic cysteine protease. The calpastatin gene, unlike ldhc, is the product of alternative promoter usage by which a truncated testis specific isoform of the somatic calpastatin is produced. Testis calpastatin (tCAST) is transcribed and translated in round spermatids. The promoter region and coding exon is located within an intron of the somatic gene. We have co-localized the testis calpastatin and calpain to the region of the sperm between the plasma membrane and outer acrosomal membrane where presumably it may be a player in the events associated with the acrosome reaction and/or with sperm-egg fusion.

A third example is UDP-N-acetylhexosamine pyrophosphorylase, described originally as AgX, the product of an alternatively spliced mRNA. A 16 amino acid deletion in the protein product results in a change in substrate specificity.

The large number of testis specific and testis abundant isozyme and protein isoforms suggests that they are not a biological curiosity, but rather are required for both full and complete spermatogenesis and for sperm function. Mechanisms regulating testis-specific gene expression, and structure/function aspects of testis gene expression will be addressed in this report.

Senile plaques consist of beta-amyloid (Aβ), and is the major pathology found with Alzheimer's Disease (AD). Aβ is particularly sensitive to oxidation, but can also produce reactive oxygen species (ROS) during Aβ-fibril formation. Cells from AD subjects are more sensitive to oxidation than non-AD age-matched controls, and it appears that a number of proteins are preferentially oxidized in plasma samples from AD compared to non-AD. We are using immunoprobes specific for oxidized proteins to elucidate the mechanism of oxidative damage and apoptosis in the neuron and to evaluate the potential of oxidized isoforms as biomarkers for early detection of AD.

The first batch of transgenic fish was produced by microinjection of human growth hormone gene into fish eggs and then a model of transgenic fish was established. In comparing with the control fish, the transgenics not only grew faster but also were more efficient in utilizing dietary protein. The transgenics had significantly higher body contents of dry matter and protein, but lower of lipid. Two groups of mice, fed with “all-fish”-transgenics or the control fish, did not show any significant differences in physiological and pathological characteristics. In addition, the genetic and the ecological safety of transgenics are also evaluated.

Aldehyde dehydrogenase (ALDH) isozymes, ALDH1 and ALDH3, and albumin, are the major soluble proteins within human corneal and lens epithelial cells. These ALDHs may perform a variety of functions in human anterior eye tissues: the oxidation of UVR-induced peroxidic aldehydes; the maintenance of high levels of reduced coenzyme with these cells; serving as ‘crystallin’ proteins to assist in the transmission of visible light; and in the biological filtration of UV-B radiation. It also appears likely that NAD(P)H contributes strongly to the absorption of UV-A and UV-B by corneal and lens epithelial cells.

Eutherian mammals utilize random X-chromosome inactivation during embryogenesis to achieve dosage compensation between XX female and XY male somatic cells. However, during gametogenesis the single X becomes inactivated in spermatocytes and both X chromosomes become active in oocytes. The function of these changes in X-chromosome activity in germ cells remains unclear. Marsupials use non-random inactivation of the paternal X to achieve dosage compensation in somatic cells. These observations are consistent with recent molecular analyses regarding the mechanism of X-chromosome inactivation in eutherians, and support the hypothesis that changes in X chromosome activity observed during gametogenesis in eutherians represent evolutionary remnants of an earlier mammalian dosage compensation mechanism which still operates in marsupials.

The major unresolved problem haunting evolutionary genetics is how much of genome organization and genetic diversity at the protein, DNA, and chromosomal levels is adaptive, processed by natural selection, and contribute to differential fitness? Rephrased, what is the relative importance of selective (natural selection) and seemingly nonselective (mutation pressure in the broadest sense, recombination, migration and genetic drift) factors in genome evolution? Since 1974 the Institute of Evolution at the University of Haifa has tested genetic diversity and spatiotemporal divergence in diverse and numerous natural populations of plants, fungi, and animals at local, regional, and global scales. The results indicate that protein and DNA diversities are heavily structured, and vary nonrandomly in all populations, positively correlated with ecological heterogeneity and stress, and are often negatively correlated with population size. Natural selection appears to be the main evolutionary driving force, overriding other forces in orienting evolution. Neutrality and near-neutrality theories of molecular evolution are rejected by the evidence as prime movers of evolution. Spatiotemporal abiotic and biotic environmental heterogeneity and stress can orient genome architecture and maintain genetic diversity in nature. Modern molecular ecological genomics can highlight the evolution of genetic polymorphism and genome evolution.

Endothelial nitric oxide synthase (eNOS) is constitutively expressed and produces constant NO output responsible for the basal vascular tone and vascular wall remodelling. Reduced NO production contributes significantly to vascular diseases. Sequence variants in the eNOS gene were associated with coronary atherosclerosis, hypertension, myocardial infarction and stroke in some studies but not in others. While frequency distributions of the eNOS sequence variations differ considerably among different ethnic populations, the genotype-phenotype associations appear to be cigarette smoking dependent. We suggest that certain genotype related CAD risk is conditional on the coexistence of a specific adverse environmental factor.

A gene-for-gene phylogeny approach has been used to study the evolution of the central pathways of carbohydrate metabolism distributed between chloroplasts, the cytosol, mitochondria and glyoxysomes in higher plants: the Calvin cycle, glycolysis/gluconeogenesis, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle. In general, the gene trees support the view that the nuclear genes for Calvin cycle enzymes were acquired from the cyanobacterial progenitors of plastids and were transferred to the nucleus.

But genes for two Calvin cycle appear to have originated from α-proteobacterial (mitochondrial) progenitors, the encoded products having been routed to a new organelle subsequent to gene transfer. The genes for glycolytic/gluconeogenetic enzymes in the cytosol also seem to be acquisitions from organellar genomes. Most TCA cycle genes originated from α-proteobacterial progenitors, with two notable exceptions that seem to represent inheritances from archaebacteria. The genes for glyoxylate cycle enzymes show marked proteobacterial affinities, whereby some have originated through duplications of preexisting genes for mitochondrial isoenzymes. Overall, the trees underscore the role of gene transfer, gene duplication and gene diversification in the evolution of compartmentalized metabolism in higher plants.

In many plant-pathogen interactions resistance to disease is determined by the interaction of plant resistance (R) genes and pathogen avirulence (Avr) genes. To determine the molecular basis of pathogen perception by plants, we study the interaction between tomato and the leaf mould pathogen Cladosporium fulvum. Tomato Cf genes confer resistance to C. fulvum infection through recognition of distinct Avr proteins. Cf proteins are predicted extracellular membrane-anchored leucine-rich repeat (LRR) glycoprotein receptors. Cf loci are genetically complex and genes from the same locus encode proteins that are more than 90% identical. Most amino acid sequence differences correspond to putative solvent-exposed residues within a conserved structural motif of LRR proteins that determines recognition specificity. Sequence analysis of Cf gene loci has also provided insight to the molecular mechanisms that generate R gene diversity.

Nitrogen heterocyclic estrogen analogs first synthesized in our laboratory [1] competitively inhibit 17β-hydroxysteroid dehydrogenase from human placenta [2]. The 16,17-fused ring pyrazole estrogen analog I forms strong steroid-to-protein hydrogen bonds at the enzyme's steroid-binding site. Key analogs were similarly tested with estrogen receptors in a gene expression system, employing luciferase reporter genes in HeLa cells transfected with human estrogen receptor-α (ERα) or rat estrogen receptor-β (ERβ). Employing ERα or ERβ receptor complexes of the analogs as mediators of gene expression, versus estradiol or a pure anti-estrogen, ICI-164,384, showed the analogs to be weak estrogens but not antiestrogens.

Many Asia people possess a variant form of liver mitochondrial aldehyde dehydrogenase where a lysine replaces a glutamate at position 487 in the 500 amino acid homotetrameric enzyme. From the three-dimensional structure of the enzyme, it appeared that residue 487 interacts with two arginine residues, 264 in the same subunit and 475 in a different one. We used site directed mutagenesis to probe for why the Oriental variant had a high Km for NAD and a low specific activity. The results show that these interactions are not the sole reason for the altered properties of the Oriental variant.

Self-incompatibility (SI) is an important intraspecific reproductive barrier to prevent self-fertilization in flowering plants. In many cases, SI is controlled by a single multi-allelic locus, the S locus. Molecular analysis of self-incompatible species of the Solanaceae, Scrophulariaceae and Rosaceae have shown that a class of ribonucleases encoded by the S locus, known as S RNases, determine the stylar expression of SI but not its pollen expression. A different gene is thought to control pollen expression of SI (pollen S gene). Here, we present some progress made towards molecular cloning of the pollen S in Antirrhinum using two approaches, S-locus directed transposon tagging and map-based cloning. Possible pathways of how S RNases interact with pollen S gene product to achieve self and non-self pollen recognition are discussed.

Specific sulphonamide inhibitors of carbonic anhydrase also inhibit the growth, and suppress the invasion, of certain types of cancer cells in culture, suggesting potential for cancer therapy. Inhibition of cell growth may be mediated through a reduction in bicarbonate provision by the cytosolic CA II and mitochondrial CA V isozymes, for the synthesis of nucleotides and other cell components. It is hypothesized that suppression of invasive properties may be mediated through inhibition of the cancer-associated, cell surface isozymes, CA IX and CA XII, resulting in a less acidic extracellular pH. CA IX may be a useful marker for renal clear cell and cervical carcinomas and a valuable adjunct to PAP screening. CA XII may be a useful marker for colorectal tumours.

A typical bioanalytical system usually consists of three classical steps, i.e., sample preparation, chemical reaction and detection. The total integration of these three steps has been the dream for many years for both academic researchers and entrepreneur. The marriage between molecular biology and the semiconductor industry for the first time brings hope to the scientific community. This presentation will describe the efforts towards the construction of microchip-based total analytical system or laboratory-on-a-chip. Progress made on microscale separation and isolation of cells, DNA amplification (PCR or strand displacement amplification) in microchips, and detection of specific sequence information on chips (via either chip-based capillary electrophoresis or electronic hybridization) will be presented.

Functional genomics can be described as scientific and technological approaches which are being applied to bridge genomic research with the discovery and development of disease-relevant therapeutic targets. These scientific approaches can offer significant opportunities in the search for causal and disease modifying therapies to better treat society's most outstanding medical needs. The use of functional genomic approaches is demonstrated in our recent efforts to elucidate cellular events leading to tumor cell cycle arrest in response to the inhibition of histone deacetylase, an important regulator of gene transcription.

In 1970, Wu and Kabat proposed an algorithm to calculate the variability of a specific site, defined by the number of different amino acids of a given position divided by the frequency of the most common amino acid of the site. This algorithm is then widely applied to MHC and TCR systems to understand their polymorphism and their relationship to diseases. Consider that the Wu-Kabat and it's modified index are not sensitive enough to evaluate polymorphism contributed by scarcely appeared members in a set of entities, and are excessively sensitive to one or two of the most common members, we propose a new algorithm to evaluate the variability of a given site with greatly improved accuracy. This new index is applied to HLA-DRB1 sequences to make further understanding of this gene.,predicting that residues 9 to 13 and residue 31,33 may correlate with antibody binding of HLA-DRB molecule which is well documented.

Transvection is an allelic interaction mediated by homologous chromosome pairing. Such interactions can modify gene expression. One gene that displays transvection effects is yellow of Drosophila melanogaster, as indicated by intra-allelic complementation between certain alleles. Transvection at yellow were originally documented using the y² mutation which is caused by the insertion of a gypsy retrotransposon. To understand the molecular mechanisms involved in transvection and investigate whether gypsy can enhance transvection at yellow, we screened through a collection of tissue-specific yellow mutations. We identified one allele that is mutant in wing and body pigmentation as y². Furthermore, we found that this allele can transvect with both y² and yellow null alleles (y^null) carrying promoter disruptions. To determine the molecular basis for these unusual complementation properties, we characterized this new allele. Our results demonstrate that this mutation is caused by a large insertion in the 5' regulatory region. Molecular characterization shows that the insert does not contain gypsy element, suggesting that this transposable element is not required for transvection at yellow. In addition, our results indicate that both cis and trans enhancers may play a role in transvection.

Trophoblasts synthesize dominant negative transacting factors that suppress expression of MHC genes. We report our initial experience in cloning a class II suppressor. A human trophoblast cDNA library was transfected into HeLa cells that were then stimulated with IFN-γ. Low class II expressors were isolated by flow cytometry. Individual low expressors were expanded by limited dilution. cDNA from clones that remained class II negative contained a 0.48 Kb non-coding RNA. Cross species activity was tested in murine B cells. This showed 80 to 90% suppression of class II antigen and mRNA.

The null activity mutants of phenoloxidase, Mox^GM95 and Dox-3^KD95, in Drosophila melanogaster were characterized by native polyacrylamide gel gel electrophoresis. cDNA fragment that is coding MoxGM95 was determined by RT-PCR from 10 g third-instar larvae as the starting material. The results of cDNA sequencing analyses, minimum size of the translated region was 1,011 and number of deduced amino acid residues was 337. The 74 insertion sequence, included stop codon, was found at the catalytic site of prophenoloxidase. The functional significance of this mutation was discussed.

Today molecular information systems are available that integrate different molecular database systems. However, the electronic information system KEGG represents the Biochemical Pathways and allows the access to different database systems which show the static representation of the molecular data and knowledge. The next important step is to implement molecular information systems which will allow to integrate different molecular database systems and analysis tools. In our paper we present an Integrative Molecular Information System for the simulation of metabolic networks.

Restriction analysis of genomic DNA from five mouse strains revealed previously unknown variations of the mouse TNAP gene, i.e. point mutations and intron-size differences. Our cloning and sequencing of 17.5 kb of the Balb/c TNAP gene allowed for a detailed analysis of these polymorphisms. Comparing the exonic sequences with previously published cDNAs, we found amino acid substitutions compatible with protein polymorphisms observed electrophoretically. Most variations, however, are due to intronic sequence polymorphisms, including variable length simple sequence repeats.

Arachidonic acid (AA), the most abundant polyunsaturated fatty acid in mammalian systems is oxygenated in rat testis mainly by two pathways - the cyclooxygenase (COX) pathway leading to the formation of prostaglandin F_2α and the lipoxygenase (LOX) pathway to produce mainly 12-HETE in the seminiferous tubules and 5-HETE in Leydig cells. LOX activity is found to be present both in the cytosol and microsomes with cytosol having thrice the activity compared with that of microsomes. Studies on the COX isozymes revealed the constitutive expression of COX-2, an inducible form expressed in response to inflammatory and mitogenic stimuli, in the spermatogonia of the seminiferous tubules. COX-2 cDNA from rat testis was synthesized by RT-PCR using primers based on 5' and 3' ends of the published human sequence to clone and further characterize COX-2 in rat testis.

The Fang Zheng silver crucian carp is a kind of bisexual triploid fish which can bring forth gynogenesis under induction of allogynogenetic fish sperm. The F1 generation of it, obtained under induction of sperm of wild carp, is called allogynogenetic silver crucian carp. The event that the heterogeneous fish sperm can also bring forth numerous biological effects in the filial generation, as it induces gynogenesis in fish, is called effect of heterogeneous sperm (heterogeneous sperm effect). RAPD was applied with 154 random primers in analyses of polymorphism in population of Fang Zheng silver crucian carp (♀), wild carp (♂) and their filial generation, allogynogenetic silver crucian carp. The electrophoretic bands related to of heterogeneous sperms effects were sought and used as probes after cloning, for Southern blot hybridization with the amplification products and genomes of above-mentioned tested fish. The results indicated that there exists a highly homogeneous fragment in the genomes of allogynogenetic crucian carp and wild carp, which is lacking in Fang Zheng silver crucian carp. It indicated the possible hybridization between the genome of the DNA fragments of male and female nuclei during fertilization. It provides a possible mechanism for the effect of heterogeneous sperm.

A regulated developmental system of carrot was established and was used to study gene expression during somatic embryogenesis. An improved cDNA representational difference analysis (RDA) was developed to isolate somatic embryogenesis related genes. ABA was thought as an important factor in sucrose signal transduction after endogenous ABA levels of carrot somatic embryos were assayed under different sucrose concentration in MS media and deregulated culture. A new cDNA fragment of LEA gene induced by ABA was obtained from carrot somatic embryo under regulated state. The result supports that the regulated-deregulated cultivation of carrot somatic embryo is similar to the dormancy –germination process of seeds.

DNA methylation is an epigenetic genome-wide general regulatory mechanism that affects qualitatively and quantitatively the expression of many genes and has been considered important for the manifestation of heterosis. DNA methylation in maize was found to be genotype, tissue and developmental stage specific. Growth conditions affected the level and pattern of DNA methylation. Our studies indicated that hybrids were less methylated than their parental inbreds and remained less methylated under stress. These findings support the hypothesis that selection of inbreds may lead to gradual accumulation of methylated sites, which could be released and/or re-patterned when the lines are crossed to generate hybrids.

Although O₂ is essential for aerobic life, when reduced, it produces unstable reactive oxygen species (ROS) that are highly toxic and, if unabated, can lead to severe physiological dysfunctions and cell death. At certain cellular concentrations, ROS are also known to play roles as second messengers in signal transduction pathways in both plant and animal cells. ROS are formed during normal metabolism and under extreme biotic and abiotic environmental conditions. Antioxidant enzymes play a key role in protecting cells against oxidative damage by scavenging and/or modulating ROS levels. However, the underlying mechanisms by which antioxidant genes perceive environmental cues to effect the antioxidant defense system are as yet not understood. The maize antioxidant defense system has been extensively investigated in our laboratory, and recent results suggest that component genes are significantly induced by many environmental factors, directly or indirectly, related to oxidative stress. Our studies indicate that the observed changes in antioxidant gene expression are likely caused by stress-mediated metabolic alterations leading to elevated ROS levels, and that each gene promoter contains multiple regulatory motifs to perceive different stress signals at different developmental stages and under oxidative stress. Hence, the maize antioxidant defense system is an ideal model to decipher the mechanisms by which these important genes are regulated to perceive external cues to effect a response by eukaryotes against the ravages of oxidative stress.

The following sections are included:

• INDEX