Continuation of Lecture 14, and Full 15

Bioinformatics

Interdisciplinary field that uses computer science, mathematics, and statistics to analyze complex biological datasets, such as DNA, RNA, and protein sequences.

Importance of Bioinformatics

Crucial for comparing and analyzing DNA/RNA/Protein sequences, creating databases, and facilitating biological research.

Annotation

identify gene regulatory elements using bioinformatics tools

GenBank – NCBI

-Largest publicly available genomic database. Acquires data from databases in Japan and Europe.

-Scientists deposit sequence data to get accession numbers for easy access and retrieval.

BLAST

-software used to compare a segment of genomic DNA to known sequences in a huge databases.

-Identifies portions that align with or are the same as existing sequences.

-only works if a similar gene sequence is already in the database.

E value

-Expect Value

-Based on number of matching sequences in database expected by chance.

Lower E value

-(closer to 0) indicate higher significance of the match. this match is not due to chance.

Higher E value

-indicate significant match. This could be due to chance.

Gene Prediction software program

-Gene prediction software uses signals like start/stop codons and regulatory sequences to guess where coding regions (exons) and non-coding regions (introns) are in DNA.

Open Reading Frames (ORF’s)

-Protein-encoding gene

-An ORF is just a sequence of triplet nucleotides that is translated into an Amino Acid of a protein.

-Typically begins with ATG.

-Ends with TAA, TAG, TGA

Genetic Testing application; Human Genome Project

-Gene-based technologies impact disease diagnosis and treatments.

-Human Genome Project advances accelerate gene identification for diseases and traits, enabling specific pharmaceuticals.

Noninvasive prenatal genetic testing

Cell-free DNA (cfDNA): DNA fragments from dead and dying cells circulate in the bloodstream.

Fetal DNA in maternal blood: Approximately 3- 6% of the DNA in a pregnant mother's blood is from the fetus.

BabySeq Project

-The BabySeq Project sequences newborn DNA to identify genetic disease risks early and improve preventive healthcare.

It can help find changes that cause health risks.

Single Gene disorder detection from SNP’s

-single-letter DNA changes (SNPs) can reveal mutations and identify whether someone is a carrier or affected by a genetic disorder.

Aneuploidy Detection

-Using chromosome copy number to detect aneuploidy.

Genetic testing for cats and dogs

-Dogs: Wisdom Panel, Embark

-Cats: Wisdom Panel, Basepaws

Gives you information on ancestry, breed identification, traits, disease risk. BUT raises ethical concerns for databreach and bankruptcy.

Genomic Wide Association studies (GWAS)

-Analyze genomes of large populations to identify genes influencing disease risk.

-Study diseases and traits like autism, obesity, diabetes, cancers, schizophrenia, and intelligence.

-Compare genomes of individuals with and without a disease.

-Identify genetic variations (e.g., SNPs, CNVs) associated with disease risk.

-Statistical Analysis: Predicts the impact of genetic variations on disease development.

Methods to locate Quantitative trait locus related to the complex trait

-Linkage association

-Genome wide association

Linkage association

-Uses a pedigree

-Mutation identical by descent. Association between genetic markers and phenotypes.

-tracks inheritance patterns in families to identify DNA regions shared among affected individuals, helping locate genes responsible for single-gene traits.

Genome-wide Association (GWAS)

-Population based.

-GWAS looks across many unrelated people to find DNA markers linked to traits—especially complex traits influenced by many polygenetic factors and the environment factors.

-Associated with Mendel Inhertiance

Synthetic Biology

-Design new organisms with useful functions, such as microbes, to provide clean energy or break down toxic wastes

-Study the minimal genome to identify essential genes for life, to then create new organisms.

Specification versus Determination versus Differentiation

Specification: The plan. The cell is leaning toward a fate, but can still change

Determination: Commitment. The cell is locked into its fate. Gene activity is fixed.

Differentiation: Realization. The cell becomes what it is supposed to be, develops structure and function.

Intrinsic versus Extrinsic

Intrinsic: Inside the cell. Information is passed down during cell division.

Extrinsic: Information is received from cells surroundings.

Coordinated Molecular Regulation

DNA methylation, histone modification, transcription factors, and RNA molecules all work together to regulate gene expression and hence a cells fate.

-coordinated molecular regulation does NOT alter the underlying DNA sequence

MZT= maternal to zygotic transition

-Also known as the Embryonic Genome Activation stage.

-Transcription begins during preimplantation development.

-Mouse: 2 cell stage

-Humans: 4 cell stage

-Bovine: 4-8 cell stage

-Development of the embryo before MZT depends on mRNA and proteins in the unfertilized egg. Sperm have little mRNA and proteins.

Blastocyst

-Consists of trophectoderm (TE) and inner cell mass (ICM).

-TE is also present in the late blastocyst, but the ICM has segregated into two lineages: epiblast (EPI), which gives 3 germ layers, and primitive endoderm (PE), forming the yolk sac for nutrient and support.

Developmental arrest

Developmental arrest from maternal control to embryonic control by transcriptional inhibitors

3 Germ layers

-Ectoderm (Outer Layer): Epidermal cells of skin, Central Nervous System, Pigment cells.

-Mesoderm (Middle Layer): Notochord, Bone, Kidney, blood cells, muscle, connective tissue, circulatory system.

-Endoderm (Inner Layer): Gives rise to the epithelial lining of the gastrointestinal and respiratory tracts, as well as organs like the liver and pancreas

Germ cells

primordial germ cells – PGCs, are destined to form sperm or eggs and are separated from somatic cells very early, often before or during early gastrulation.

Totipotent Cell

-At fertilization, an egg and sperm fuse to form a single totipotent cell, the zygote, up to 8 cell • Totipotent Cells can become any cell type, including the placenta.

Pluripotent

-Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst.

-These cells are pluripotent cells and can form any of the 200+ different cell types in an adult, but are not able to form a placenta

-Self-renew: Proliferate indefinitely

-Differentiate into various cell types in 3 germ layers

Multipotent Stem cells

-Can differentiate into a limited range of cell types, usually restricted to a specific tissue or lineage

Unipotent

-The least flexible stem cells; can only differentiate into one specific cell type, but possess the ability to self-renew

DNA methylation and genomic imprinting life cycles

-Mammalian cells go through two full cycles of demethylation. There is no evidence that epigenetic information can survive two rounds of this biochemical cleansing.

-Somatic cells: Maintains imprints throughout life.

-Germ cells:Imprints are erased in the primordial germ cells

-Genomic imprints:re-established on the DNA in male or female gametes for maternal or paternal imprints

-In a new embryo, the imprints are maintained during embryonic development, erased again in the germ cells

Epigenetic reprogramming

important for establishing and maintaining cell identity during development.

DNA methylation and histone modifications are extensively erased and then re-established across the genome. reprogramming can reverse cells back to a more flexible (stem-like) state.

Erasure and reprogramming

Erasure and reprogramming of epigenetic marks are normal parts of the mammalian life cycle.

demethylation cycles in early embryogenesis

-Paternal genome (blue): is initially actively demethylated by the TET3

-Maternal genome (red): demethylation is solely passive due to DNMT1 dilution, hence the sharper demethylation slope for the paternal curve.

-zygotic paternal and maternal pronuclei undergo global demethylation during the pre-implantation stages, except for imprinted germ cell differentiated methylated regions (gDMRs), which are maintained via DNMT1 activity.

-After implantation, the blastocyst acquires de novo methylation patterns catalyzed by DNMT3A and DNMT3B

Stem cell Therapy

-Regenerative medicine • Drug discovery • Stem cell Therapy

-Ethical concern because they are destroying embryos.

-Immune system rejection between donor and recipient.

-Induced pluripotent stem cells can solve the immune system rejection problem.

-Convert differentiated cells into pluripotent cells or another cell type (direct reprogramming)

-The ultimate goal of generating the reprogrammed cell is to use them for regenerative medicine to restore structurally and functionally damaged tissues and organs.

Evolutionary Conservation

-can be studied using model organisms.

-The size and shape of all animal bodies are controlled by a common set of genes and developmental mechanisms.

-Species variation is the result of different patterns of expression of highly conserved regulatory genes, such as the homeotic (abbreviated as Hox) genes, and not by species-specific genes

Early Drosophila embryogenesis

Early Drosophila embryos divide their nuclei many times WITHOUT forming separate cells.

Patterning and Morphogenesis

Patterning: cells are spatially organized into a blueprint.

Morphogenesis: cells subsequently transform into structured, 3D tissues and organs.

Body Plan Formation

-establishing an organism’s spatial organization, symmetry, germ layers, and segmentation.

-The primary body axes, anterior-posterior (front-totail) and dorsal-ventral (top-to-bottom), are established early through maternal genes.

-Tightly regulated by 1)Maternal effect genes- sets up axis2)segmentation genes-divide body into segments 3)homeotic selector genes- assigns identity to each segment.

Maternal effect genes

-Stored in unfertilized egg

-Positional gradients known as morphogens

-regulate the expression of zygotic genes.

Anterior Posterior Axis

-Anterior system (head): Bicoid (Bcd), Hunchback (Hb)

-Posterior system (tail): Nanos (Nos), Caudal (Cad)

The Bicoid gene encodes the morphogen responsible for head structure in Drosophila.

(All of them will act as morphogens except for nanos)

-These are also all maternal effect genes.

Segmentation genes

-Transcribed in nuclei of developing embryos in response to the distribution of maternal effect proteins

1. Gap genes divide the embryo into broad regions (head, thorax, abdomen).

2. Pair-rule genes further subdivide the embryo into smaller regions, about two segments wide. 3. Segment polarity genes divide each segment into anterior and posterior regions.

-Deleterious recessive mutations in homozygous form lead to embryonic lethality

Homogametic genes

-Homeotic (Hox) selector genes determine the action of maternal and zygotic segmentation genes, specifying which adult structures (body parts) will form from each segment.

Homeosis

-transformation of one body part into another.

-Mutation causes one body segment to be transformed into another.

- Phenotypes can result from either loss-offunction or gain-of-function mutations.

-Encode transcription factors with a highly conserved DNA binding domain (homeodomain)

-Regionally expressed along the antero-posterior axis • Control body patterning in all animals

- first discovered in Drosophila

Antennapedia complex

gives head and thoracic segments T1 and T2 their identity

Bithorax complex

gives T3 and abdominal segments their identity

Antennapedia

-Inappropriate expression of Antennapedia in the head converts Antennae Into Legs

-Gain-of-function mutation,

-Chromosomal inversion brings a gene under the control of promoters of other genes.

The Colinearity Rule

-Homeotic Gene Expression.

- The physical order of the genes in the complex is the same as the spatial and temporal order of their expression along the anterior-posterior axis.

-3’ genes are expressed earlier and more anteriorly than 5’ genes.

Ultrabithorax

-Loss-of-Function Mutations.

-Result in a Four-winged Fruit Fly.

-Produced by mutations in enhancer sequences for Ultrabithorax gene (Ubx)

-Thoracic segment 3 (T3) converted into another thoracic segment 2 (T2)

-Typically, posterior Hox genes inhibit anterior Hox genes. When Ubx is mutated, Antp transforms T3 to T2.

-Anterior transformation: If a posterior gene is removed, the segment might take on a more anterior identity, or if an anterior gene is missing, it may be replaced by even more anterior structures.

Summary of Patterning and Morphogenesis in embryogenesis

1. The combined action of maternal-effect genes and zygotic segmentation genes defines the embryo's anterior-posterior axis, segment number, size, and polarity.

• Transcription factors play a key role in activating these genes in a sequential manner, ensuring proper segmentation and development.

• Certain genes in the zygote’s genome are activated or repressed according to a positional gradient of maternal gene products.

2. Expression of three sets of segmentation genes divides the embryo into a series of segments along its anterior–posterior axis.

• These segmentation genes are transcribed in normally developing embryos, and mutations of these genes have embryonic-lethal phenotypes.

3. Both segment polarity genes and Hox genes control the differentiation of each segment of the future larva.

Evolutionary Conservation of Homeotic Gene Organization

-Hox genes play a master role in the development of the anteroposterior axis in various multicellular organisms, including a wide variety of animal taxa and most tunicates.

-Vertebrates have 4 clusters of Hox genes (HOXA, HOXB, HOXC, and HOXD)

(Orthologs vs. Paralogs)

• Orthologs: Genes closely related to each other in different species thought to be derived from one common ancestral gene ✓ e.g. lab in Drosophila is orthologous with a1, b1, and d1 in mouse

• Paralogs: Genes having similar structures, the same relative position on each of the four chromosomes, and similar expression patterns. Caused by gene duplication within 1 species. ✓ a1, b1 and d1 are paralogous in mouse

Real Examples of Hox Gene Expression in the Mouse

-In Situ Hybridization: mapping the spatial and temporal expression of Hox genes, which pattern the anterior-posterior axis during embryonic development. -Use labeled RNA or DNA probes complementary to HOX mRNA.

-The anterior boundary of expression correlates with a gene’s position on the chromosome.

Hox Gene Expression Along the Antero-posterior axis of the Mouse Mesoderm

-Functional redundancy between members of a paralogous group (such as a1, b1 or a4, b4) can explain mild phenotypes when individual genes are knocked out.

Comparison of Vertebral Patterns and Hox Gene Expression in the Chick and Mouse

There is a good correlation between anatomy and Hox gene expression.

More Dramatic Transformations Are Observed When All Members of a Paralogous Group are Knocked Out

ICM

ICM splits into TWO lineages:

1. Epiblast (EPI) → forms the entire embryo (all 3 germ layers)

2. Primitive endoderm (PE) → forms yolk sac / support structures