Creation of a Synthetic Organism with the Smallest Complement of Genes

The tools available to molecular biologists especially in regard to gene sequencing and assembly allow investigators to produce nucleotide sequences that incorporate specific genes and gene clusters into DNA created in-situ.  A team of investigators headed by Craig Venter from the J. Craig Venter Institute in La Jolla California in effect created a microorganism containing 473 genes (Syn 3.0).  The purpose of this investigation was to determine the minimal amount of genetic material required to sustain life as an autonomous organism and successfully reproduce. 

In 2010, Venter and his colleagues created an entire chromosome from the bacterium, Mycoplasma mycoides (this organism has only one chromosome) and demonstrated that this synthetic chromosome was completely functional.  They did this by stripping out the naturally occurring DNA from the mycoplasma, M. capricolum and replacing it with the synthetic chromosome.  The modified organism was called Syn 1.0 and with its complement of 901 genes was shown to be completely viable and capable of reproduction.

With this material in hand, the investigative group sought to assemble Syn 3.0 by methodically whittling down the DNA in Syn 1.0 to the smallest number of genes required to sustain life. The result of this painstaking work was Syn 3.0

What makes this current result so remarkable is that this organism is entirely new.  Of the 473 genes, 149 (31.5%) are of unknown function; therefore, additional work will focus on the discovery of the function of these apparently essential genes.  Syn 3.0 may prove to be an invaluable tool in understanding the evolution of life on planet earth.

What is the A1C Test?

The disease diabetes mellitus occurs in two different forms – juvenile or adult-onset.  In either case, the source of the illness is lack of or reduced production of the hormone insulin (see image below) whose role is to enhance the uptake of glucose circulating in the blood by tissue cells, especially adipose and skeletal muscle.  Insulin (as seen below) is referred to as a globular protein

Insulin is normally produced by specialized beta cells resident in the Islets of Langerhans within the pancreas.  It has been established that juvenile diabetes is an auto-immune disease in which the immune system of the patient attacks these beta cells.  Adult-onset diabetes, on the other hand, has a strong association with obesity.

This inability to transfer glucose to tissue cells where it is utilized for energy, leads to high concentrations of glucose in the blood (hyperglycemia).  Over a prolonged period of time, this hyperglycemic state results in very serious and ultimately life-threatening complications including blindness, impaired kidney function, cardiovascular issues leading to heart trouble and leaving victims prone to amputation.  These deleterious side effects arise as the excess glucose in the blood reacts with proteins in various tissues throughout the body – this biochemical reaction is referred to as glycosylation.

Once the cause of diabetes was discovered, it was realized that an obvious therapeutic approach is to give the patient insulin from an external source.  Before the advent of DNA recombinant technology, patients were given insulin harvested from cow pancreas (bovine insulin).  Bovine and human insulin are close enough in structure to allow bovine insulin to have an efficacious effect.  Currently, of course, human insulin is readily available.

Over many years of treating diabetic patients with human insulin, it was realized that periodic administration of insulin is not comparable to the body’s finely tuned regulation of insulin production so as to maintain optimal levels of blood glucose.  In response to this deficit, a technology arose to employ the use of an insulin pump in order to maintain a steady stream of insulin along with careful monitoring of blood glucose levels.  In addition, considerable emphasis has been placed on nutrition, exercise and weight control especially in regard to adult-onset diabetes.

Another important tool in the treatment of diabetes is the so-called A1C test.  This test provides information as to the average level of blood glucose over a 3-month time span; it is also referred to as the glycohemoglobin test.  This test measures the amount of glycosylated hemoglobin.  Hemoglobin is the specialized protein in red blood cells that is designed to carry oxygen to the tissues.  This protein reacts with excess glucose in the blood.  Since red blood cells are recycled by the body over a 3-month period, the test provides information about the average level of glucose in the blood over this period of time.  The result of this test is reported as a percentage.  The normal value is about 5.7%.  The higher the percentage, the higher the blood glucose level has been over the past 3 months.

The A1C test is an important tool in determining the efficacy of treatment for diabetic patients.  

The Role of the Large Protein Titin in Dilated Cardiomyopathy

There is a serious heart condition referred to as dilated cardiomyopathy (DCM) that is a major cause of heart failure and often results in premature death; this disease is found in one in two-hundred and fifty adults (0.4%).  DCM can originate either as a result of an underlying vascular problem or can have a genetic origin.  This report will focus on the progress that has been made in regards to the genetic implications of this condition.

Through the work of Dr. John T. Hinson at the Division of Cardiovascular Medicine at the Brigham and Women’s Hospital, Boston MA and his colleagues from many diverse institutions, it has been show that mutations of a large protein that constitutes one-half of the sarcomere (a structural unit of a myofibril in striated muscle) are the most common cause for DCM.  This protein is referred to as titin (TTN) (see image below) and the mutations involved result in a truncated version of TTN.  These genetic variants of TTN are referred to as TTN-truncating variants (TTNtvs).

Since the functional significance of TTN within the muscle sarcomere was unclear, the investigators involved in this research, applied the tools of molecular biology to better define the role played by TTN within heart muscle.  To accomplish this, they grew out cardiac micro-tissue cultures comprised of cardiomyocytes derived from pluripotent stem cells (iPS) that were harvested from the patients studied.  When these patient-derived cells were compared to those derived from normal individuals (the controls), it was discovered that, “certain missense mutations like TTNtvs diminish contractile performance and are pathogenic.”

Furthermore, these patient-derived cardiomyocytes also demonstrated sarcomere insufficiency, reduced responses to mechanical and biochemical stress as well as impairment in critical cell signaling pathways.  All of these results, when taken together, point to TTNtvs as playing a causative role in genetically-induced DCM.  This is a significant finding with broad implications.

Molecular Disease

There are a host of diseases that are a direct result of a mutation in a single gene. Examples of this kind of disease are many including sickle cell anemia, severe combined immunodeficiency disease (SCID) and many others. The world famous chemist, Linus Pauling (1901-1994) coined the term Molecular Disease to refer to this type of illness. He was awarded the Nobel Prize in Chemistry for his work in 1954.

 We shall use sickle cell anemia as a case in point to illustrate how a singular change in the molecular structure of a gene can have profound consequences for the organism. A patient with sickle cell anemia presents the following symptoms often beginning at 4 months old:

• Painful episodes that can last hours or days Attacks of abdominal pain 
• Bone pain 
• Breathlessness 
• Delayed growth and puberty 
• Fatigue 
• Fever 
• Jaundice 
• Paleness 
• Rapid heart rate 
• Ulcers on the lower legs (in adolescents and adults). 

Other symptoms include:

• Chest pain 
• Excessive thirst 
• Frequent urination 
• Painful and prolonged erection (priapism - occurs in 10 - 40% of men with the disease) 
• Poor eyesight/blindness 
• Strokes 
• Skin ulcers. 

This disease was thought to have a genetic etiology based upon the epidemiological data which showed its prevalence among individuals of African descent (one in twelve African Americans are heterozygous for this trait). Furthermore, these data also pointed to a recessive trait i.e. both alleles have to possess the altered gene for the symptoms to appear.

The disease presents with a singular characteristic – misshapen red blood cells (See Figure 1). This change in morphology from the normal disc-shaped cell to crescent-shaped is a direct result of the altered tertiary structure of the hemoglobin molecule (referred to as Hemoglobin S). Normal hemoglobin has a globular tertiary structure (See Figure 2).

Figure 1 

Figure 2   

Hemoglobin is the protein found in circulating red blood cells (RBC) that is responsible for transporting oxygen through the blood stream and ultimately to all the tissues of the body. This change in the structure of hemoglobin is a direct result of a replacement of one of its constituent amino acids. In this case the sixth amino acid in the protein polypeptide chain has been changed from glutamic acid to valine. This singular modification causes the deoxygenated form of the protein to clump together (See Figure 3). This replacement results in a molecule that is no longer readily soluble in the cell cytoplasm of RBC. As a result of this one change, the overall morphology of RBC is changed from the normal disc shape to crescent shape and they can no longer flow readily through the bloodstream as shown in Figure 1.

 Figure 3   

Since the sequence of all proteins is determined by the specific sequence of nucleotides in the genes responsible for their production, sickle cell anemia is a direct result of a single point mutation in the gene carrying the information for the production of hemoglobin found on human chromosome number 16 – hence the term, molecular disease.

 Since this disease has such a serious impact on the mortality for those who are afflicted, it might be asked why natural selection has not deleted this deleterious gene over the course of evolution. There is an explanation for this seeming contradiction. Those individuals that are heterozygous for this trait – where only one of the alleles has the mutation – have a subset of RBC that is misshapen. The remaining cells are normal, and these individuals are symptom free. Those who are homozygous for the trait – where both alleles have the mutation – have the full-blown disease. It appears that those who are heterozygous for the trait are protected from the malaria parasite whose host is RBC. Therefore, natural selection would favor those who are heterozygous for sickle cell anemia (carriers). 

In the past, this type of illness has been impervious to the possibility of a cure, since its origin resides in the very makeup of an individual’s heredity as expressed through the genes. However, since, red blood cells originate in the bone marrow, sickle cell anemia can be cured by bone marrow transplantation, but this approach has its own set of significant risks.

Recent advances in molecular biology and gene therapy have demonstrated that this daunting limitation can be effectively breached using gene therapy. SCID is a particularly devastating and ultimately fatal disease in which the affected child has no defense against infections. Through the ground breaking work of Dr. Alessandro Aiuti, ten patients suffering from SCID are still alive. The mutated gene in this condition is the ADA gene responsible for the production of the enzyme adenosine deaminase - ADA. The laboratory of Dr. Aiuti from the San Raffaele Institute for Gene Therapy in Milan successfully used the following procedure: bone marrow cells from the patients involved were incubated with a specially engineered virus containing the normal ADA gene. These engineered cells were reintroduced into the patients. Positive results were seen almost immediately following treatment. A similar approach has been used in the treatment of a disease characterized by a congenital degeneration of the retina. In this study four of six patients had a notable improvement of vision.

The latest advance has been made with Adrenoleukodystophy (ALD), a disease linked to the X chromosome. This is a severe neurodegenerative disease that leads to destruction of myelin, the outer membrane of nerve cells, in the brain and severe nervous system dysfunction. This disease is caused by a mutation in the ABCD1 gene. The first successful clinical test using gene therapy for ALD has recently been reported by Dr. Nathalie Cartier and her colleagues from the University of Paris-Descartes in Paris, France. The approach used was to take hematopoietic stem cells (HCS) from two young male patients and incubate their cells with a virus that was engineered to carry the normal ABCD1 gene. These modified cells were then reintroduced to the patients. Eventually, blood cells with the normal gene were found distributed throughout each patient’s body. Within 14 to 16 months post treatment, cerebral demyelination was arrested and neurological and cognitive functions remained stable. The patients’ own cells were used in this procedure; this avoids any need for a donor and obviates any concern of possible rejection. This is an extraordinary result and has profound implications for the future of gene therapy in medicine and may eventually find application in the treatment of sickle cell anemia.

Viruses Shown to Transfer Immunity between Infected Cells

Interferons (IFN) are known to play a critical role in the innate immune response to viral infection.  The molecular mechanism responsible for eliciting this response has been well studied.  It has been shown that IFN production is initiated by signaling pathways activated by molecular sensors in the presence of viral particles including cytosolic DNA sensors.  One of these DNA sensors is Cyclic GMP-AMP synthase (cGAS).  This enzyme, when activated, catalyzes the synthesis of a second messenger referred to as cyclic GMP-AMP (cGAMP).  This messenger subsequently activates transcription factors that “turn on” the genes responsible for the production of IFN (STING).

This cascading sequence of steps occurs within the infected cell.  This activation of IFN may also be spread to nearby cells connected via a gap junction.  However, Dr. A. Bridgeman and his colleagues from the medical Research Council Human Immunology Unit at the Medical Research Council Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, suspected that this immunity could also be transferred by the viral vector to whatever additional cells it was infecting.  They proposed that the infecting virus might actually incorporate and transfer the cGAMP second messenger.  This suggestion has some precedence in that the human immunodeficiency virus 1 (HIV-1) has been shown to incorporate host-derived substances.  Given this known behavior, the investigators hypothesized that cGAMP could be picked up by infecting virions, incorporated and subsequently passed on to additional host cells and in that way actually inadvertently spread the immune response.

In order to test this proposal, they used modified and attenuated virions to infect a human embryonic kidney cell line that expressed STING and that were able to induce the production of IFN in response to cGAMP.  When these same viruses were subsequently exposed to a target cell line, they were shown to have induced the expression of IFN suggesting that their working hypothesis was correct.  The investigators painstakingly ruled out other possible variables that might explain this phenomenon.  Even virus-like particles stripped of their RNA genome still induced IFN production in target cells.   Finally, they went on to demonstrate unambiguously that cGAMP was, in fact, packaged into the virus studied.

This is an interesting finding that may have clinical value.  In light of this evidence, the authors suggest that “using viral vectors with cGAMP therefore holds promise for vaccine development.”  

The Genomics of Mental Illness

Mental illness covers a wide range of diseases including schizophrenia (SCZ), autism spectrum disorder (ASD), chronic depression and bipolar disorder (BPD).  The aberrant behavior associated with these disorders has long been ascribed to factors other than that of a genetic or organic origin.  On account of the tremendous strides that have been made in the fields of molecular biology and human genetics, there is a new understanding of the role of human genes in the development of psychiatric diseases.

Daniel H. Geschwind and Jonathan Flint from the Department of Neurology, Psychiatry and Human Genetics at the David Geffen School of Medicine in University of California in Los Angeles have published a review article in the Journal Science (Vol 349, No 625, pp 1489-1493) in which they describe the current scientific understanding of the role genes play in mental illness.  Within the body of this review, they make a number of salient points.

Due to remarkable technological advances, variations at millions of Single Nucleotide Polymorphisms (SNPs) within the human genome can be detected.  Furthermore, with the use of microarrays, genome-wide association studies (GWASs) can be performed that can establish associations between disease states and common genetic alleles.  As a result of such exhaustive studies, it appears that GWASs generally lie within regulatory regions of the genome.  Since regulatory sites usually lie within close proximity to the genes that are regulated, it is not unreasonable to assume that it is such functional genes that are affected.

 

In addition, microarrays have also identified copy number variants (CNVs) associated with both SCZ and ASD.  These CNVs are the result of either a gain or loss of DNA involving DNA segments > 1 kilobase (kb) in size.   Another area of intense investigation involves genomic sequencing that focuses on the complete protein coding sequence also referred to as whole-exome sequencing (WES).  WES reveals the DNA sequences that have the coding information within the entire genome for all the proteins destined for production.  To date, tens of thousands of individuals have been analyzed in this way.  From this extensive data, rare protein variants have been shown to be associated with SCZ and ASD.

Although the application of these methodologies have contributed greatly to the understanding of the role of genes in mental illness, the various mental illness disease states appear to involve a multiplicity of genetic loci making it difficult to pinpoint the precise etiology of the disease process.  However, great progress continues to be made in this area of research, making it more likely that complete molecular mechanisms will eventually be uncovered.   

The Role of ADAR in Mammalian Development

Adenosine deaminase enzymes that act upon RNA (ADAR) have been shown to play a critical role in mammalian development.  ADAR is responsible for the conversion of adenosine to inosine in double-stranded RNA (dsRNA).

                    

Adenosine

Inosine

                                                          

Literally hundreds of thousands of these transforming events have been reported within human cells.  This editing appears to occur predominantly in noncoding repetitive elements within the genome.  Additionally, there are three forms of ADAR that have been identified – ADAR1, ADAR2 and ADAR3.  They seem to be widely expressed during early development especially in the embryonic and post-natal development period.  To date, of the three forms of ADAR, only ADAR1 and ADAR2 have been shown to demonstrate enzymatic activity on natural substrates in vitro.

Using the mouse animal model, ADAR2 has been shown to modify an essential receptor in the brain.  Experimental animals who die from seizures have successfully rescued when a genomic substitution mimicked the adenosine to inosine modification of the RNA transcript.  This result proved to be an elegant demonstration of the mode of action of ADAR2 in vivo.

The role of ADAR1 within the living organism remained unknown.  This prompted Dr. Brian J. Liddicoat and his colleagues from the Department of Medicine at St. Vincent’s Hospital University of Melbourne, Australia to engage in an elaborate experimental investigation to uncover its role.

The results of their extensive efforts demonstrated that, “The A-to-I editing of ADAR1 is essential for embryonic development and the maintenance of hematopoiesis in vivo.”  Hematopoiesis refers to the production of viable circulating blood cells.

This is an important finding and is a significant contribution to the overall understanding of the complexities and intricacies of mammalian development.