Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Yellow mutant albinism: cytochemical, ultrastructural, and genetic characterization suggesting multiple allelism.

This report describes three sisters, including monozygotic (MZ) twins, with clinical, ultrastructural, and histochemical features typical of yellow mutant albinism; This form of albinism is clinically similar to the tyrosinase-positive type, but hair bulbs showed (1) organelles similar to red hair pheomelanosomes and (2) absence of tyrosinase activity. Classical tyrosinase-negative albinism was found in a maternal cousin of the probands. Pedigree analysis of this family suggests multiple alleles occupying a single locus.     

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.     

What can we learn from noncoding regions of similarity between genomes?

Background  

In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.     

Daytime Blue Light Enhances the Nighttime Circadian Melatonin Inhibition of Human Prostate Cancer Growth

Light controls pineal melatonin production and temporally coordinates circadian rhythms of metabolism and physiology in normal and neoplastic tissues. We previously showed that peak circulating nocturnal melatonin levels were 7-fold higher after daytime spectral transmittance of white light through blue-tinted (compared with clear) rodent cages. Here, we tested the hypothesis that daytime blue-light amplification of nocturnal melatonin enhances the inhibition of metabolism, signaling activity, and growth of prostate cancer xenografts. Compared with male nude rats housed in clear cages under a 12:12-h light:dark cycle, rats in blue-tinted cages (with increased transmittance of 462–484 nm and decreased red light greater than 640 nm) evinced over 6-fold higher peak plasma melatonin levels at middark phase (time, 2400), whereas midlight-phase levels (1200) were low (less than 3 pg/mL) in both groups. Circadian rhythms of arterial plasma levels of linoleic acid, glucose, lactic acid, pO₂, pCO₂, insulin, leptin, and corticosterone were disrupted in rats in blue cages as compared with the corresponding entrained rhythms in clear-caged rats. After implantation with tissue-isolated PC3 human prostate cancer xenografts, tumor latency-to-onset of growth and growth rates were markedly delayed, and tumor cAMP levels, uptake–metabolism of linoleic acid, aerobic glycolysis (Warburg effect), and growth signaling activities were reduced in rats in blue compared with clear cages. These data show that the amplification of nighttime melatonin levels by exposing nude rats to blue light during the daytime significantly reduces human prostate cancer metabolic, signaling, and proliferative activities.Abbreviations: A-V, arterial–venous difference, ipRGC, intrinsically photosensitive retinal ganglion cell, LA, linoleic acid, 13-HODE, 13-hydroxyoctadecadienoic acid, TFA, total fatty acidsLight profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in all mammals and is essential to life on our planet.^{2,15,28, 40} The light–dark cycle entrains the master biologic clock, located in the suprachiasmatic nucleus of the brain, in an intensity-, duration-, and wavelength-dependent manner.^8-13 Photobiologic responses, including circadian rhythms of metabolism and physiology, are mediated by organic molecules called ‘chromophores,’ which are contained within a small subset of retinal cells, called the intrinsically sensitive retinal ganglion cells (ipRGC).^{16,29,31,36,41,49,53,59} In humans and rodents light quanta are detected by the chromophore melanopsin, which detects light quanta in principally the short-wavelength, blue-appearing portion of the spectrum (446 to 477 nm), and transmits its photic information via the retinohypothalamic tract to the ‘molecular clock’ of the suprachiasmatic nucleus. This region of the brain regulates the daily pineal gland production of the circadian neurohormone melatonin (N-acetyl-5-methoxytryptamine), which results in high levels produced at night and low levels during daytime.^38,54 The daily, rhythmic melatonin signal provides temporal coordination of normal behavioral and physiologic functions including chronobiologic rhythms of locomotor activity,² sleep-wake cycle,^2,14 dietary and water intake,^2,51 hormone secretion and metabolism.^5,44,47,61 Alterations in light intensity, duration, and spectral quality at a given time of day,^{8-13,17,19-22,24,61} such as occurs in night-shift workers exposed to light at night,^26,34,46,57 acutely suppresses endogenous melatonin levels in most mammalian species^{9,11,44,45,54,55} and may lead to various disease states, including metabolic syndrome^5,61 and carcinogenesis.^4-7,17,18Recent studies from our laboratory^{5,20,23-25,60,61} have demonstrated that relatively small changes in the spectral transmittance (color) of light passing through translucent amber (>590 nm), blue (>480 nm), and red-tinted (>640 nm) polycarbonate laboratory rodent cages, compared with standard polycarbonate clear cages (390 to 700 nm), during the light phase markedly influenced the normal nighttime melatonin signal and disrupted temporal coordination of metabolism and physiology.^19,24,61 Most notable was our discovery that, in both male and female pigmented nude rats maintained in blue-tinted rodent cages, nighttime melatonin levels were as much as 7 times higher than normal nighttime peak levels in animals maintained in all other cage types.¹⁹ An earlier study in human subjects diagnosed with midwinter insomnia coupled with low nighttime melatonin levels demonstrated that daily exposure to intense morning bright polychromatic light therapy for up to one week resulted in a restoration of nocturnal melatonin levels to those of control subjects.³⁵ In another study, exposure to blue-tinted (470 nm) LED light (100 lx) for approximately 20 min in the morning after 2 sleep-restricted (6 h) nights led to earlier onset of the melatonin surge at nighttime.³⁰In the United States alone this year, approximately 240,000 men will be diagnosed with prostate cancer, and nearly 30,000 will die from this disease (National Cancer Institute; www.cancer.gov/). Epidemiologic studies have shown that night shift work, which involves circadian disruption, including nocturnal melatonin suppression, markedly increases prostate cancer risk in men.^{26,34,46,57,58} Both in vitro and in vivo studies have demonstrated that melatonin inhibits human prostate cancer growth, including that of androgen-receptor–negative, castration-resistant PC3 human prostate cancer cells.^20,29,42,56 Cancer cells depend primarily on aerobic glycolysis (Warburg effect) over oxidative phosphorylation to meet their bioenergetic needs supporting biomass formation.⁵ The Warburg effect is characterized by increased cellular uptake of glucose and production of lactate despite an abundance of oxygen. Investigations have shown that signal transduction pathways that include AKT, MEK, NFκB, GS3Kβ, and PDK1 drive the Warburg effect.^5,61 In addition, cancer cells rely on increased uptake of the ω6 fatty acid linoleic acid (LA), which is prevalent in the western diet.^4-6 In most cancers, LA uptake occurs through a cAMP-dependent transport mechanism, and LA is metabolized to the mitogenic agent 13-hydroxyoctadecadienoic acid (13-HODE). In most tumors, 13-HODE plays an important role in enhancing downstream phosphorylation of ERK 1/2, AKT, and activation of the Warburg effect, thereby leading to increased cell proliferation and tumor growth.^4-6 Melatonin, the principal neurohormone of the pineal gland and whose production is regulated by the suprachiasmatic nucleus,^4,5 modulates processes of tumor initiation, progression, and growth in vivo.⁵ The circadian nocturnal melatonin signal not only inhibits LA uptake and metabolism, the Warburg effect in human cancer xenografts, and ultimately tumor growth, but it actually drives circadian rhythms in tumor metabolism, signal transduction activity, and cell proliferation. These effects are extinguished when melatonin production is suppressed by light exposure at night.⁵In the present investigation, we examined the hypothesis that the spectral transmittance (color) of short-wavelength (480 nm) bright light passing through blue-tinted standard laboratory rodent cages during the light phase not only amplifies the normal circadian nocturnal melatonin signal but also enhances the inhibition of the metabolism, signaling activity, and growth progression of human PC3 androgen-receptor–negative human prostate cancer xenografts in male nude rats.