Protein stability

• The lifetime length of different proteins can vary from just a few minutes to years. Protein stability is a critical component of gene regulation. There are several systems that play roles in destroying proteins.
  • The ubiqutin-mediated proteolysis pathway is found in all eukaryotic organisms. This system modifies proteins by attaching onto them the 76 amino acid protein "ubiquitin," which is highly conserved in all organisms (thus is ubiquitous). A protein thus modified ('ubiquitinated') is quickly shuttled into a multiprotein complex that can be described as a 'garbage disposal' (actually it's called the proteasome). The proteasome digests ubiquitinated proteins, removing them from the cell.
  • The amino terminal amino acid also plays a role in protein stability. The is called the "N-end rule," which just means that some amino acids in that position can cause a protein be remarkably unstable, while other amino acids in that position can give a protein a long half-life.

Genetic control of development in Drosophila

Drosophila is a model system because of its shirt generation time, small size, and simple genetics, with just four chromosome pairs. Because insects are complex multicellular organisms, the fruit fly has been used as a genetic model to study development of morphological traits in complex organisms, as well as many other aspects of eukaryotic functions. If you are curious about fly genetics, check out: The InterActive Fly webpage: http://sdb.bio.purdue.edu/fly/aimain/1aahome.htm

Drosophila researchers have many resources to draw from, as a result of hundreds of laboratories that have worked on this system for almost a century. These resources include genetic maps with hundreds of mapped mutations, and access to stocks of adult flies that carry most of these mutations. The genes in the organism are easily obtained from vast clone libraries that include all of the genes in the organism, most of which are available to researchers upon request. Finally, the entire genomic sequence has been determined, so all genes in the organism are known; these data are easily accessed on the internet.

Many studies in Drosophila have tried to dissect the mechanisms that control development of the fly. We’ll talk about some of these, and about how mechanisms of gene regulation control formation of the embryo from an egg, as well as how an adult it formed from an embryo.

Embryo development in Drosophila (Fig 17.25-26)

• Syncitial phase of development (embryo cell division movies:http://www.biology.ucsc.edu/people/sullivan/images.html)
• Cellularization
• Differentiation in the larva, formation of imaginal disks
• Pupation and adult development

Gradients define the fly embryo

• Most of the cells in the embryo differentiate according to their positions on the embryo, which is determined based on the relative concentration of dorsal-ventral (D-V) and anterior-posterior (A-P) gradients. These gradients cause the parts of the embryo to adopt different fates, and form distinct body segments on the developing fly.
• An A-P gradient determines features that cause the head end of the fly to be different from the posterior end. Though the book focuses on genes involved in anterior-posterior development, we won't cover those here or on the exams. Instead, we'll focus on regulation of dorsal-ventral polarity. However, realize that both type of gradients (D-V and A-P) combine to specify most part of the fly embryos.

Determination of Dorsal-Ventral polarity in the developing embryo (this pathway is not discussed in the book, so refer to these notes)

• A dorsal-ventral gradient cause the back of the fly to develop differently from the belly.
• This involves the dorsal pathway, named because most mutations in this pathway cause the embryo to develop an abnormal dorsalized appearance where the belly and back do not differ (this pathway is not discussed in the book, so refer to these notes)
• The extracellular ligand Spatzle is produced predominantly in the ventral region of the fly embryo
• The presence of Spatzle is detected by Toll receptors on the cell surface
• Binding of Spatzle to the Toll receptor causes activation of a protein kinase called Pelle, located in the cytoplasm
• Activated Pelle then phosphorylates Cactus, which is a protein that binds to the transcription factor Dorsal. This binding keeps Dorsal localized in the cytoplasm, which prevents the transcription factor from affecting gene expression
  Cactus, once phosphorylated becomes ubiquitinated
• Ubiquitinated Cactus is rapidly proteolyzed, thus liberating the bound transcription factor Dorsal.
  Dorsal then migrates to the nucleus, where it binds to certain gene promoters and activates transcription of many genes
• This transcription causes the ventral part of the embryo to differentiate ventral features. Thus, through Dl pathways gene products, the extracellular ligand (Spatzle) triggeres changes in gene expression in the nucleus. Because Spatzle is concentrated on the ventral part of the embryo, this provides a means for regulating expression of ventral identity genes.
  

The Dorsal Group Genes

• Mutations in any of about 11 genes would produce a fly that produced defective embryos that exhibited dorsalized features, thus the naming of the pathway (Notes on the genes).
  • A. Easter, Produces the D-V asymmetry in ligand with a higher concentration of Ea in ventral part of embryo,
  • B. Spätzle, the asymmetrically accumulated ligand,
  • C. Toll, a transmembrane receptor,
  • D. Tube, a mysterious protein that is needed for coupling of the receptor to the effector kinase,
  • E. Pelle, the effector protein kinase,
  • F. Cactus, a cytoplasmic 'sequesterase' that binds Dl.
  • G. Dorsal, a transcription factor,
  • H. Various target genes give rise to the differences between dorsal and ventral cell type (target gene examples):
    • Various target genes are expressed in the ventral portion of the embryo
    • Disruption of this regulation can cause flies to develop 'upside-down,' in a sense, with dorsal-localized cells exhibiting ventral-cell-like features.

     

Regulation of the dorsal-ventral signal transduction pathway (Click on Genes for Notes)

Phenotypes of Drosophila Mutants

• Null mutations in all of the genes in the dorsal-ventral group (A-G), except cactus, produce a dorsalized phenotype, in that ventral structures fail to differentiate.
• Mutations in Cactus produces a ventralized phenotype.
• Epistasis analysis has permited the ordering these genes into a pathway.

Different parts of the embryo percieve their position based on both dorsal-ventral and anterior-posterior gradients. This information determines the identities of the different segment on the late stage embryo-adult. Specific clusters of cells in the mature embryo form "imaginal disks" that will provide the cells in metamorphosis that produce eyes, legs, wings, and other organs in the adult fly (Fig 17.29)

After the segment identities have been determined, the action of homeotic genes comes into play. These specify the types of structures that each segments will form. Some of these include appendages such as legs, wings and antennae.

Mutations in homeotic genes can cause inappropriate formation of appendages, showing that single genes can control the fate of large anatomical structures.

Examples of homeotic genes include:

• Genes in the Antennapedia complex (ANT-C) affect development of structures on the head of the fly and two thoracic segments. Most mutations of these genes are lethal. Mutations of one, called Antennapedia, is not lethal and when mutated causes leg structures to form in place of antennae (Fig. 17.31).
• The Bithorax complex (BX-C) of genes similarly effect development of posterior segments of the fly, namely the third thoracic segment and the abdominal segments. Most mutations of genes in this region are lethal, though some produce viable offspring, including the Ubx mutation that causes halteres to develop into wings, giving the flies an abnormal second pair of wings (Fig. 17.30)

Upon sequencing of genes within the NT-C and BX-C, a curious conserved region of 180 bp coding sequence was observed, which codes for a 60 amino acid segment conserved in the genes in each complex. This region in the DNA of these genes is called a “homeobox” and the encoded conserved 60 amino acid segment of each protein a “homedomain.” Homeotic genes are also called "Hox genes"

The homedomain portion of these proteins binds to DNA in the promoters of target genes, and thus regulates the activity of those genes. Thus many genes in the Antennapedia and Bithorax complexes, act as transcription factors, and regulate expression of many other genes that are important in organ development.

Oddly, the linear sequence of homeotic genes in Drosophila are arranged in a linear sequence that corresponds to the sequence of structures along the fly body. For example BX-C genes that effect wing formation are located 5’ to genes affecting posterior abdominal segment identity (Fig. 17.32)

Hox genes have been found in many animals havin ga segmented developemtnal pattern, including insects, vertebrates, of course humans. The linear order of these genes and their function along the axis of the organism is a common feature, called the "colinearity rule."

What effect does modification by ubiquitination of cellular proteins have on those proteins?

For the following questions, refer to the lecture notes:

What is the main defect in embryo development in flies that carry mutations in most Dorsal pathway genes?

How is embryo development affected in in flies that carry mutations in the Cactus gene?

What is the role of ubiquitination on Cactus function?

What function is provided by Dorsal?

How is Dorsal's activity regulated by Cactus?

What is the effect of mutations within homeotic genes?

How to study for the Final Exam:

The questions we will have asked on the three trimester exams were designed to test concepts we felt were important, amd thus we will draw heavily on the concepts covered in those questions when we prepare the Final Exam. So understand the problems on the three exams and their solutions, and you will be well prepared for the final. We won't write the precise same questions, but the concepts tested will be very similar. There will also be four lectures that were not covered on the three midtem exams, so there will also be questions from that material too.

Botany 132 Homepage:  http://www.uvm.edu/~bot132/ For questions about this site: TPD