Evolution Of New Functions Research Articles

Analysis of large libraries of protein mutants using flow cytometry

Publisher Summary This chapter introduces a flow cytometry, as an extremely useful tool for the quantitative analysis of protein libraries, and for the isolation and characterization of very rare clones. Flow cytometry is a one-dimensional implementation of fluorescence microscopy, where the particles or cells under observation flow in a stream past a light source and optical collection apparatus. Flow cytometers are designed to be easily programmed to collect cells of the desired optical properties, such as light scattering or fluorescence profile, using either a closed cycle or an electrostatic droplet deflection technology. The screening of protein libraries by flow cytometry offers the following advantages: quantitative assays can be performed on large populations, with single cell resolution; and multiple quantitative parameters for each cell can be analyzed simultaneously, including various fluorescence signals and forward, and 90u light scattering. Flow cytometry represents the most advanced library screening tool, in terms of instrument sophistication, throughput, and the degree of enrichment of positive cells. The quantitative nature of flow cytometry makes it an ideal tool for the exploration of protein sequence space and for the evolution of new function.

Evolution of gene families

In eukaryote genomes, there are many kinds of gene families. Gene duplication and conversion are sources of the evolution of gene families, including those with uniform members and those with diverse functions. Population genetics theory on identity coefficients among gene members of a gene family shows that the balance between diversification by mutation, and homogenization by unequal crossing over and gene conversion, is important. Also, evolution of new functions is due to gene duplication followed by differentiation. Positive selection is necessary for the evolution of novel functions. However, many examples of current gene families suggest that both drift and selection are at work on their evolution.

Preservation of Duplicate Genes by Complementary, Degenerative Mutations

The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow 1996(p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh 1995 (p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff 1997 (p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. 1997 (p. 362)

Determining the evolutionary potential of a gene.

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base-substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta-galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.

Pathways of ubiquitin conjugation.

The covalent attachment of the polypeptide ubiquitin to proteins marks them for degradation by the ubiquitin/26S proteasome-dependent degradation pathway. This pathway functions in regulating many fundamental processes required for cell viability. Phylogenetic analysis of ubiquitin sequences reveals greater variability among lower eukaryotes and defines essential residues, many of which are conserved among the three ubiquitin-like proteins known to undergo parallel ligation pathways. The hierarchical design of the ubiquitin conjugation mechanism provides great flexibility for the divergent evolution of new functions mediated by this posttranslational modification. Within this hierarchy, a single ubiquitin-activating enzyme provides charged intermediates to multiple targeting pathways defined by cognate ubiquitin carrier protein (E2)/ligase (E3) pairs. Sequence analysis of E2 isozymes shows that the E2 superfamily is composed of distinct function-specific families. The apparent lack of E2/E3 specificity suggested in the literature results from the presence of multiple isozymes within many E2 families and erroneous family assignments based on incomplete data sets. Other apparent inconsistencies are explained by interfamily sequence relationships among some E2 isoforms.

Myosin heavy chain gene amplification as a suppressor mutation in Caenorhabditis elegans.

In the nematode, Caenorhabditis elegans, the body wall muscles contain paramyosin and two different types of myosin heavy chain, MHC A and MHC B. In mutants that do not express MHC B or that express defective paramyosin, muscle structure is disrupted and movement is impaired. Second site mutations in the sup-3 locus partially reverse these defects and are correlated with a 2- to 3-fold increase in the accumulation of the MHC A isoform. The sup-3 mutations occur at a high frequency (10(-4] after ethyl methanesulfonate (EMS) mutagenesis. This is comparable to the average EMS-induced mutation rate per gene in C. elegans. In this paper we show that the sup-3 mutation is an amplification of the structural gene for the MHC A protein, myo-3. We employed genomic Southern hybridization with MHC gene-specific probes in order to measure the copy number of the myo-3 gene relative to that of the MHC B gene, unc-54. We have identified the putative amplification junctions for these sup-3 alleles using a set of cosmid clones which encompass myo-3 region. Although it has been suggested that gene amplification plays an important role in evolution, there are few known cases of gene amplification in the germ line cells of multicellular organisms. The results shown here provide a clear example of a heritable gene amplification event that occurs at a high frequency in the germ line. Similar events may thus represent the initial event in the evolution of new function and in the formation of multigene families.

HOW COURTS LOOK AT NURSE PRACTICE ACTS

fter taking their state boards, few nurses think about their license to practice except when the time comes to renew it. a surprise, then, when the family member you couldn't get along with files a complaint with the board of nursing. So what? you think. What are they going to do? Take away my license? Actually, in some states, the board of nursing could use just those grounds for a cause summons-a summons for you to appear before the board to show why your license should not be revoked or suspended. In Alaska's revised nursing practice act, for example, unprofessional conduct is listed as grounds for loss of license. So, while the statutory language of state nursing practice acts, at first blush, may seem less than exciting, the acts are invaluable reading because of their importance in defining and regulating your But the real meat of what nurses can do under the law is often found in the nursing board's rules and regulations. Although these are not law, they have the force of law. Board rulings, a third source for determining the scope of nursing in your state, have no force of law. Nevertheless, before you embark on a gray area of practice, you might want to ask the board if it has issued (or will issue) an advisory opinion or ruling. Advisory opinions are rulings formulated by nursing boards, often in response to a formal request by a nurse or facility for clarification of a clinical practice activity. In addition, the board may submit a question to a specific state agency or department such as the Attorney General's department. Advisory opinions from the Attorney General's office are not law but can be persuasive in a judicial proceeding. As nursing and medical activities increasingly overlap, courts are being petitioned to interpret challenged sections of nurse practice.acts. In one of the most important cases dealing with advanced nursing practice thus far, the Missouri Supreme Court reviewed the (1975) nursing statute in light of the old law.1 The case went to court when the Board of Registration for the Healing Arts (medical board) threatened to sue nurse practitioners in a family planning clinic for the alleged unauthorized practice of medicine. The nurses were doing breast and pelvic exams, lab tests, and birth control counseling and were providing birth control products. When the court looked at the nurse practice act to determine whether the nurses were practicing within the scope of nursing, it found an open-ended definition of professional nursing introduced by the phrase including, but not limited to... . The court said that the use of this language evidences an intent to avoid statutory constraints on the evolution of new functions for nurses delivering health services. When the court compared the existing act with the earlier act, it noted that the requirement for a physician to directly supervise nursing functions had been eliminated. This was interpreted as a legislative desire to expand the scope of authorized nursing practice. More commonly, a court is called on to interpret the nursing practice act when a dispute arises from a disciplinary action or negligence suit. In Massachusetts, the board of nursing suspended an RN's license for practicing as a nurse midwife without credentials.2 The nurse, Janet Leigh, argued that she was engaged in lay midwifery, not nurse midwifery. Massachusetts has no statutory prohibition against lay midwifery. Leigh, who graduated from a college nursing program in 1969, practiced predominantly in obstetrics. She testified that she had begun to practice as a lay midwife in 1977 and had delivered more than 600 infants, including home births. She maintained her nursing license and did a little private duty, but never sought approval from the board to practice as a nurse midwife. The incident that occasioned the disciplinary action involved the death of an infant, whose umbilical cord prolapsed during a home delivery. Leigh called an ambulance to take the mother to a hospital where Leigh had back-up physician coverage. Leigh's hand was in the woman's vagina and she was trying to hold back the cord. The ambulance crew announced that they would take over and did not want Leigh in the ambulance. The ambulance personnel testified that after Leigh said she was a registered nurse they let her accompany the patient in the ambulance. The baby was strangled by the cord. The court found Leigh guilty not because she practiced lay midwifery, but because she violated the nursing practice law and board regulations. The question before the board became whether the nurse's conduct was a violation of the nursing practice statute or whether the nurse was merely practicing lay midwifery. The board could not impose sanctions if the nurse was practicing lay midwifery, since the court held that such midwife practice by non-

Directed evolution of cellobiose utilization in Escherichia coli K12.

The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in evolution of new functions. Escherichia coli does not use beta-glucoside sugars; however, mutations in several loci can activate the cryptic bgl operon and permit growth on the beta-glucoside sugars arbutin and salicin. Such Bgl+ mutants do not use cellobiose, which is the most common beta-glucoside in nature. We have isolated a Cel+ (cellobiose-utilizing) mutant from a Bgl+ mutant of E. coli K12. The Cel+ mutant grows well on cellobiose, arbutin, and salicin. Genes for utilization of these beta-glucosides are located at 37.8 min on the E. coli map. The genes of the bgl operon are not involved in cellobiose utilization. Introduction of a deletion covering bgl does not affect the ability to utilize cellobiose, arbutin, or salicin, indicating that the new Cel+ genes provide all three functions. Spontaneous cellobiose negative mutants also become arbutin and salicin negative. Analysis of beta-glucoside positive revertants of these mutants indicates that there are separate loci for utilization of each of the beta-glucoside sugars. The genes are closely linked and may be activated from a single locus. A fourth gene at an unknown location increases the growth rate on cellobiose. The cel genes constitute a second cryptic system for beta-glucoside utilization in E. coli K12.

MUTATION-SELECTION BALANCE IN MULTI-LOCUS SYSTEMS. I. DUPLICATE GENE ACTION

A theoretical model is presented that extends the case of selection against homozygous recessives counterbalanced by mutation to a system of n loci. This extension allows analysis of the role of gene duplication in the evolution of new function. The aspect of retention of function for sufficiently long periods of time to allow for divergence vs. silencing of nonfunctional loci is discussed in relation to examples in salmonid and catastomid fishes and in the globin-like clusters.

On the evolution of new metabolic functions in diploid organisms.

Evolution of lactose utilization via the ebg system of Escherichia coli requires both structural gene (ebgA) and regulatory gene (ebgR) mutations. Because evolution of new metabolic functions in diploids might be subject to constraints not present in haploid organisms, merodiploid strains carrying a wild-type and an evolved ebgA allele, or a wild-type and an evolved ebgR allele were constructed. I show that heterozygosity at ebgA does not significantly affect the selective advantage of the evolved ebgA allele; whereas heterozygosity at ebgR eliminates the selective advantage of the evolved ebgR allele. Is is suggested that, in diploid organisms, evolution of new functions for systems under negative control would be very difficult.

Gene duplication in tetraploid fish: model for gene silencing at unlinked duplicated loci.

Several groups of fishes, including salmonids and catastomids, appear to have originated through genome duplication events. However, these two groups retain approximately 50% of the loci examined as functioning duplicates, despite the passage of 50 million years or more of mutation and selection. Although other effects are not excluded, this apparently slow rate of duplicate silencing can be explained in terms of the effects of selection against defective double homozygotes to unlinked duplicates. We have derived a computer simulation of genetic drift that affords direct evaluation of the effects of population size (N), mutation rate (micron), initial allele frequencies, back mutation, fitness, and time on the probability of fixation for null alleles at unlinked duplicate loci. The results show that this probability is approximately linearly related to population size for N greater than or equal to 10(3). Specifically, for naive populations, the time for 50% probability of gene silencing is approximately equal to 15N + micron-3/4 generations. The retention of 50% of the loci as functional duplicates may therefore result from the large effective size of salmonid and catastomid populations. The results also show that, under most conditions for populations of 2000--3000 or larger, unlinked duplicate loci will be sustained in the functional state longer than tandem (linked) duplicates and hence are available for evolution of new functions for a longer time.